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RETURN TO ISSUEPREVTechnical Note

Unambiguous Phosphosite Localization using Electron-Transfer/Higher-Energy Collision Dissociation (EThcD)

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† Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.
‡ Netherlands Proteomics Centre, Padualaan 8, 3584 CH Utrecht, The Netherlands
§ Research Institute of Molecular Pathology (IMP), Dr. Bohrgasse 7, A-1030 Vienna, Austria
∥ Institute of Molecular Biotechnology (IMBA), Vienna, Austria
*E-mail: [email protected], [email protected]
Cite this: J. Proteome Res. 2013, 12, 3, 1520–1525
Publication Date (Web):January 24, 2013
https://doi.org/10.1021/pr301130k

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Abstract

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We recently introduced a novel scheme combining electron-transfer and higher-energy collision dissociation (termed EThcD), for improved peptide ion fragmentation and identification. We reasoned that phosphosite localization, one of the major hurdles in high-throughput phosphoproteomics, could also highly benefit from the generation of such EThcD spectra. Here, we systematically assessed the impact on phosphosite localization utilizing EThcD in comparison to methods employing either ETD or HCD, respectively, using a defined synthetic phosphopeptide mixture and also using a larger data set of Ti4+-IMAC enriched phosphopeptides from a tryptic human cell line digest. In combination with a modified version of phosphoRS, we observed that in the majority of cases EThcD generated richer and more confidently identified spectra, resulting in superior phosphosite localization scores. Our data demonstrates the distinctive potential of EThcD for PTM localization, also beyond protein phosphorylation.

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Introduction

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Reversible phosphorylation of proteins is a key regulatory mechanism in living cells. (1) Protein phosphorylation can modulate protein activity, turnover, subcellular localization, complex formation, folding and degradation. Dynamic phosphorylation plays a pivotal role in almost all biological processes including cell division, differentiation, polarization and apoptosis. (2) Moreover, it is an important switch in cellular signal transduction. (3) The importance of this post-translational modification (PTM) for cell biology has driven the development of novel mass spectrometric tools for sensitive and global detection of phosphorylation. (4, 5) However, the analysis of phosphorylated peptides by mass spectrometry is still not as straightforward as for “regular”, unmodified peptides. One of the major challenges in phosphoproteomics is to improve MS level representation since phosphopeptides are usually present at substoichiometric levels. Hence, an enrichment step is necessary to enable deeper penetration of the phosphoproteome. Enrichment is typically performed by chromatography, (6) antibodies (7) or metal-ion/metal oxide affinity-based (8, 9) techniques. Two other main challenges are the identification of phosphopeptides and confident localization of the corresponding phosphosite. (10) The challenge is caused by the higher lability of the phosphate group when compared to the amide bond. A number of strategies have been proposed to circumvent poor fragmentation and improve sequence and site diagnostic fragmentation, including the use of neutral loss-triggered MS/MS/MS (11) and multistage activation (MSA) (12) in ion traps, the use of beam type CID fragmentation, (13) and electron capture/transfer dissociation (14) or a combination of some of these approaches. (9, 15)
Once phosphopeptide identification is feasible through sufficient peptide backbone fragments, it can still be challenging to pinpoint the true phosphosite. This becomes more difficult as the number of potential phosphorylation sites within the peptide sequence increases. In principle, unambiguous phosphosite localization requires site-determining fragment ions. (16) Direct validation is feasible through detection of a fragment ion that carries the phosphate group. Neutral loss fragment ions can be used as well; however, since they exhibit the same mass as a water loss from an unmodified residue they do not directly confirm the correct site. (17) Diagnostic phosphosite-specific fragments facilitate pinpointing the correct phosphosite. (18-20) Several algorithms and programs have been developed to enable automatic phosphosite localization. (3, 16, 21-26) These software tools are based on distinct but similar approaches and they all aim to provide a metric that allows for assessment of the confidence in phosphosite localization. Recently, Taus et al. have reported on a new algorithm, coined phosphoRS, (27) which presently is uniquely compatible with CID, HCD and ETD fragmentation and was optimized for both low- and high-resolution MS/MS spectra. phosphoRS provides individual localization probabilities for all potential phosphosites in a given peptide.
Generally, all scoring tools depend on the quality of the MS/MS spectra. The more site-determining ions are detected, the higher the confidence in phosphosite localization. We have recently introduced a novel fragmentation scheme combining electron-transfer and higher-energy collision dissociation, termed EThcD. (28) This method employs dual fragmentation to generate both b/y and c/z ions which leads to very fragment ion- and thus data-rich MS/MS spectra. Compared to HCD and ETD, we found a substantial increase in peptide backbone fragmentation, which translated into a remarkable average peptide sequence coverage of ∼94% for tryptic peptides. We reasoned that localization of post-translational modifications could also highly benefit from EThcD spectra. Here, we systematically assessed the impact on phosphosite localization using EThcD. In this work we evaluate the performance of EThcD in comparison to ETD and HCD using a defined synthetic phosphopeptide mixture and also on a larger data set of Ti4+-IMAC enriched phosphopeptides, all in combination with a modified version of phosphoRS.

Experimental Section

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Materials

All chemicals were purchased from Sigma-Aldrich (Steinheim, Germany) unless otherwise stated. Formic acid and ammonia were obtained from Merck (Darmstadt, Germany). Acetonitrile was purchased from Biosolve (Valkenswaard, The Netherlands).

Sample Preparation

Protein from HeLa cells was harvested and digested with trypsin, as previously described. (29) Ti4+-IMAC beads were prepared as reported elsewhere. (30, 31) Phosphopeptides were enriched as previously described. (32) Briefly, Gel-loader tips that were plugged with C8 material (3M, Zoeterwoude, The Netherlands) were filled up to 1 cm with Ti4+-IMAC beads. Columns were equilibrated with loading buffer (80% ACN, 6% TFA). Peptides were reconstituted in loading buffer, loaded onto the columns and washed with washing buffer 1 (50% ACN, 0.5% TFA, 200 mM NaCl) and subsequently washing buffer 2 (50% ACN, 0.1% TFA). Phosphopeptides were eluted with elution buffer 1 (10% NH3 in H20) followed by elution buffer 2 (80% ACN, 2% FA). Eluate was acidified and diluted with formic acid to a final acetonitrile concentration of <5%, split into three equal amounts and directly analyzed by single run LC–MS/MS utilizing ETD, HCD and EThcD, respectively.

Mass Spectrometry

All data was acquired on an ETD enabled Thermo Scientific LTQ Orbitrap Velos mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). A Thermo Scientific EASY-nLC 1000 (Thermo Fisher Scientific, Odense, Denmark) was connected to the LTQ Orbitrap Velos mass spectrometer. ETD, HCD and EThcD methods were set up as previously described. (28) Briefly, all spectra were acquired in the Orbitrap at a resolution of 7500. For HCD the normalized collision energy was set to 40%. The ETD reaction time was set to 50 ms for ETD and EThcD. Supplemental activation was enabled for ETD. HCD normalized collision energy was set to 30% for EThcD (calculation based on precursor m/z and charge state). The anion AGC target was set to 4e5 for both ETD and EThcD.

Data Analysis

Peak lists were generated using Thermo Scientific Proteome Discoverer 1.3 software (Thermo Fisher Scientific, Bremen, Germany). The nonfragment filter was used to simplify ETD spectra with the following settings: the precursor peak was removed within a 4 Da window, charged reduced precursors were removed within a 2 Da window, and neutral losses from charge reduced precursors were removed within a 2 Da window (the maximum neutral loss mass was set to 120 Da). MS/MS spectra were searched against a database containing the synthetic phosphopeptide sequences and the human Uniprot database (version v2010–12), respectively, including a list of common contaminants using SEQUEST or Mascot (Matrix Science, UK). The precursor mass tolerance was set to 10 ppm, the fragment ion mass tolerance was set to 0.02 Da. Enzyme specificity was set to Trypsin with 2 missed cleavages allowed. Data from the synthetic phosphopeptide mixture was searched with no enzyme specificity. Oxidation of methionine and phosphorylation (S,T,Y) were used as variable modification and carbamidomethylation of cysteines was set as fixed modification. Percolator (33) was used to filter the PSMs for <1% false-discovery-rate. Phosphorylation sites were localized by applying a custom version of phosphoRS (27) (v3.0 – EThcD enabled) that has been expanded to allow analysis of EThcD data. (28) Briefly, the algorithm considers both HCD- and ETD-type fragment ions at the same time. While singly and doubly charged b- and y-type fragment ions including neutral loss of phosphoric acid (H3PO4) are considered for site localization, only singly charged c-, z-radical and z-prime ions are scored.

Results and Discussion

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Increasing the confidence in phosphosite localization is a key challenge in phosphoproteomics. Site-determining fragment ions are required to unambiguously pinpoint the correct phosphosite. Observing all possible peptide backbone cleavages in a single MS/MS spectrum substantially simplifies phosphosite localization. Recently, we showed that EThcD enables complete peptide sequencing through dual fragmentation. (28) In EThcD, the peptide precursor is initially subjected to an ion/ion reaction with fluoranthene anions in a linear ion trap, which generates c- and z-ions. However, the unreacted precursor and the charge-reduced precursor remain highly abundant after ETD. In the second step HCD all-ion fragmentation is applied to all ETD derived ions. This generates b- and y-ions from the unreacted precursor and simultaneously increases the yield of c- and z-ions by fragmentation of the charge reduced precursor. Since the remaining unreacted precursor population is higher charged than the ETD-derived fragment ions one can apply a level of energy that fragments the precursor but does not induce secondary fragmentation of c- and z-ions. Here, we continue to explore the benefits of this novel fragmentation mode for the analysis of phosphopeptides.

Evaluation of Phosphosite Localization by EThcD using a Defined Phosphopeptide Mixture

To evaluate the potential added value of phosphopeptide analysis by EThcD we initially used a defined mixture of well-characterized synthetic phosphopeptides. This mixture consists of 30 phosphopeptides of varying length with up to four phosphorylated residues (see Supplementary Table 1 for a complete list, Supporting Information). We analyzed this mixture by LC–MS/MS employing ETD, HCD and EThcD fragmentation, respectively. We used identical instrument settings with the only exception being the parameters for peptide dissociation, which were set to the for each method optimized values. The data was searched with SEQUEST and the PSMs were manually validated and filtered (7 ppm peptide mass tolerance, search engine rank 1, absolute Xcorr threshold 0.4). Additionally, we considered only PSMs for which the injection time did not max out (<500 ms), that is, the target number of ions was reached. Note that this precaution was taken to exclude the number of ions as a variable that might impair the quality of fragmentation. We calculated the average precursor ion purity (PIP) (34) for each data set and found similar values, which were approximately 95% for all three techniques. Together, these stringent criteria ensure that the activation technique is the only variable that controls the fragmentation behavior. A summary of the data from this direct comparison is given in Table 1. Similar numbers of PSMs were identified for all three fragmentation techniques. We found that EThcD provided 248 PSMs while these numbers were 237 and 216 for HCD and ETD, respectively. Out of the 30 unique synthetic phosphopeptides injected ETD, HCD and EThCD identified 21, 22 and 24, respectively. We found the average SEQUEST Xcorr being highest for EThcD (2.5) followed by HCD (1.9) and ETD (1.5), which is in line with our previous results for nonmodified peptides. (28) The SEQUEST algorithm correctly annotated the known phosphosites in 79% of ETD and 78% of HCD data. Significantly, for EThcD this was over 95% (of all PSMs), which directly reflects the higher spectral quality, due to the generation of both b/y and c/z ions. This initial data suggests that EThcD provides even more extensive backbone fragmentation of phosphorylated peptides than ETD or HCD alone, facilitating sensitive phosphosite localization with very high confidence. It should be noted that the application of a site localization algorithm would be prudent for real-life samples since the true phosphorylation sites are unknown.
Table 1. Analysis of 30 Synthetic Phosphopeptides
  ETD HCD EThcD
#PSM 216 237 248
# unique peptides 21/30 22/30 24/30
average Xcorr 1.5 1.9 2.5
% PSM with correctly localized phosphosite (SEQUEST) 79% 78% 95%
# phosphosites with phosphoRS site probability >99% 478 410 423
% phosphosites with phosphoRS site probability >99% 96% 95% 97%
Recently, Taus et al. described phosphoRS, a novel tool to improve confident localization of phosphosites. (27) The software is based on validated peptide identifications provided by database search engines and calculates site probabilities for each potential phosphosite in the peptide sequence. For this study we used a modified version of phosphoRS that also enables assessment of individual phosphosite probabilities for EThcD fragmentation. We analyzed each data set using phosphoRS and found that it performs equally well for all three fragmentation techniques. Of all true phosphosites, 96% (ETD), 95% (HCD) and 97% (EThcD) were assigned a site probability >99%, which corresponds to a very high confidence in site localization (Table 1). Together, these findings suggest that EThcD generates MS/MS spectra that contain sufficient fragment ions for the unambiguous and sensitive phosphorylation site localization.

Phosphosite Localization of Ti4+-IMAC Enriched Phosphopeptides by EThcD

Next, we assessed the performance of EThcD for phosphosite localization on a larger data set. We used Ti4+-IMAC material for the enrichment of phosphopeptides from a tryptic digest of HeLa cells and analyzed equal amounts (corresponding to enriched phosphopeptides from 100 μg of protein) by LC–MS/MS with ETD, HCD and EThcD, respectively (Supplementary Figure 1A, Supporting Information). All three methods generated a similar number of MS/MS spectra. All spectra were searched with SEQUEST. The ETD data was also searched with Mascot because we found SEQUEST to perform poorly for doubly charged phosphopeptides. Note that other search engines such as OMSSA or SpectrumMill might provide larger number of identifications for ETD data. (35) However, these algorithms are currently not compatible with EThcD data and phosphoRS analysis within the Proteome Discoverer software environment. All identified PSMs were then filtered for <1% FDR using percolator to ensure consistency. In total we identified 2217 (ETD), 4179 (HCD) and 3594 (EThcD) phospho-PSMs (Table 2). Our initial analysis of a defined synthetic phosphopeptide mixture demonstrated that EThcD performs at least on the same level as HCD in terms of peptide identification. However, the overall identification success rate in the Ti4+-IMAC data set was slightly lower for EThcD compared to HCD. This can be attributed to the rigid automatic FDR filtering. The MS/MS spectra from the synthetic phosphopeptide mixture were manually validated whereas the Ti4+-IMAC data set was computationally filtered to <1% FDR. The application of EThcD, in comparison to ETD or HCD alone, significantly increases the number of fragment ions observed in the MS/MS scans. On the one hand EThcD spectra contain more sequence information, which is beneficial for inferring the peptide sequence and PTM localization. On the other hand, these additional fragment ions may also match to random peptide sequences, increasing their score and hampering the differentiation between correct and incorrect matches. Consequently, the chance for a high scoring random match will be elevated. Similar to the increased average score of decoy hits also the true hits are likely to provide on average higher scores. Depending on whether the distance between the two score distributions decreases or increases, the identification success rate will be higher or lower. Since the ID success rate is slightly lower for EThcD compared to HCD alone, the negative effect of higher-scoring random matches might be more pronounced. Thus, higher score cut-offs need to be applied in order to reach the desired FDR. A standard target-decoy approach (36) against a reversed concatenated database revealed the FDR for EThcD (2.6%) being almost twice as high compared to HCD (1.4%), which provides further evidence for this hypothesis.
Table 2. LC–MS/MS Analysis of Ti4+-IMAC Enriched Tryptic Phosphopeptides Originating from a Cellular Lysate using ETD, HCD and EThcD
  ETD HCD EThcD
#PSM 2266 4282 3679
ID success rate 25% 51% 44%
average Xcorr 1.9 2.5 3.2
% average peptide sequence coverage 83% 81% 92%
# phospho-PSM 2217 4179 3594
# phospho-sites >99% pRS probability 2002 4291 3942
% phospho-sites >99% pRS probability 81% 89% 95%
Next, we calculated the average peptide sequence coverage for all PSM. As expected, EThcD provided a substantial increase in sequence coverage (92%) compared to HCD (81%) and ETD (83%). Obtaining near-complete peptide sequence coverage tremendously simplifies phosphosite localization. We used the extended phosphoRS algorithm to validate our assumption. Remarkably, EThcD provided for 95% of all phosphosites a confident site localization probability of >99%. In the HCD data set we found that 89% of all phosphosites were assigned with a confident site localization probability >99%, while this was only 81% for ETD data set. We recalculated these number for all peptides that contain >2 residues that can be phosphorylated because singly phosphorylated peptides with only one potential phosphorylation site could bias the results toward HCD. Of all phosphosites from this subset of peptides 97% (ETcaD), 93% (EThcD) and 87% (HCD), respectively, were assigned a localization probability >99%.
For multiply phosphorylated peptides site localization becomes more challenging. Figure 1 shows an MS/MS spectrum of a doubly phosphorylated peptide upon EThcD fragmentation. The overall sequence coverage is 89% taking b/y- and c/z-ions into account. Six out of 18 amino acid bond cleavages are represented by c- and b-ions (referred to as “golden pairs” (37)). Additionally, we observed 11 z/y-ion pairs, which strengthens the argument that EThcD provides extensive sequence information that facilitates pinpointing the correct phosphorylation site. More than 95% of the phosphosites from all doubly phosphorylated peptides were assigned with a site localization probability >99%, highlighting that EThcD performs equally well with singly and doubly phosphorylated peptides. A known limitation of ETD is its inability to cleave the N–Cα bond N-terminal to proline. (38, 39) This can hamper phosphosite localization for proline-rich peptides. Generation of dual ion series in EThcD can overcome this issue. Figure 2 shows the EThcD spectrum of a singly phosphorylated peptide that contains four serine residues. The c- and z-ions derived from the ETD step cover only the N-terminal part of the peptide and the site probability is only 50%. The additional y-ions derived from the subsequent HCD activation provide supporting sequence information and cover also the two serine residues next to the prolines which enables unambiguous phosphosite localization.

Figure 1

Figure 1. EThcD MS/MS spectrum of a doubly phosphorylated peptide. RGTGQSDDSDIWDDTALIK is doubly phosphorylated and contains in total four potential phosphorylation sites. EThcD generates dual ion series that enable phosphorylation site localization with very high confidence (phosphoRS site probabilities: T(3), 0.0%; S(6), 100.0%; S(9), 100.0%; T(15), 0.0%). SEQUEST Xcorr 7.79.

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Figure 2

Figure 2. EThcD spectrum of a proline-containing phosphopeptide. This EThcD spectrum of a doubly charged peptide that contains four serine residues, one of which is phosphorylated. ETD does not cleave the N–Cα bond N-terminal to proline and the phosphorylation site probability is only 50% based on c- and z-ions alone. Dual fragmentation by EThcD generates complementary sequence information from c/z- and b/y-ions (SEQUEST Xcorr 4.10). Here, the exact phosphosite is revealed by y-ions that cover the corresponding phosphosite (phosphoRS site probabilitis: S(1): 0.0; S(3): 0.0; S(8): 99.5; S(10): 0.5). SEQUEST Xcorr 4.10.

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Conclusions

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Here we have evaluated the potential of EThcD in improving the analysis of phosphopeptides. Our data highlights the benefit of dual ion series as generated by EThcD fragmentation. We observed for a defined phosphopeptide mixture average higher SEQUEST Xcorr values, higher peptide sequence coverage and more confident phosphosite localization in EThcD compared to ETD and HCD. This finding was confirmed when we analyzed a complex phosphopeptide sample resulting from a Ti4+-IMAC enrichment of peptides from a cellular lysate. This is in line with recent reports that showed that confidence in phosphorylation site localization increases when multiple separately acquired MS/MS spectra (e.g., ETD/CID or MSA/ETD) are combined for scoring. (25, 26) For this larger data set, we observed that the identification success rate was slightly lower for EThcD compared to HCD. This can be attributed to the use of conventional database search engines that are not optimized for spectra that contain dual ion series. (40) However, the fact that both peptide sequence coverage and the percentage of localized phosphosites are higher for EThcD than for HCD suggests that once a peptide was identified, further analyses such as site localization benefit from the more data-rich EThcD spectra. In EThcD often c/b- and z/y-ion pairs are observed that increase the confidence in a particular peptide backbone cleavage. (41) We speculate that the identification success rate of EThcD for phosphopeptides can be improved by novel or optimized data analysis tools. Finally, we reason that EThcD can also be beneficial and used to improve the localization of other post-translational modifications such as ubiquitination, glycosylation or acetylation.

Supporting Information

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Additional information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

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Author Information

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  • Corresponding Authors
    • Albert J. R. Heck - Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.;  Netherlands Proteomics Centre, Padualaan 8, 3584 CH Utrecht, The Netherlands;  Email: [email protected] [email protected]
    • Shabaz Mohammed - Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.;  Netherlands Proteomics Centre, Padualaan 8, 3584 CH Utrecht, The Netherlands;  Email: [email protected] [email protected]
  • Authors
    • Christian K. Frese - Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.;  Netherlands Proteomics Centre, Padualaan 8, 3584 CH Utrecht, The Netherlands
    • Houjiang Zhou - Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.;  Netherlands Proteomics Centre, Padualaan 8, 3584 CH Utrecht, The Netherlands
    • Thomas Taus - Research Institute of Molecular Pathology (IMP), Dr. Bohrgasse 7, A-1030 Vienna, Austria
    • A. F. Maarten Altelaar - Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.;  Netherlands Proteomics Centre, Padualaan 8, 3584 CH Utrecht, The Netherlands
    • Karl Mechtler - Research Institute of Molecular Pathology (IMP), Dr. Bohrgasse 7, A-1030 Vienna, Austria;  Institute of Molecular Biotechnology (IMBA), Vienna, Austria
  • Notes
    The authors declare no competing financial interest.

Acknowledgment

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We thank Mathias Madalinski for peptide synthesis of the primeXS phosphopeptide mixture. This research was performed within the framework of the PRIME-XS project, grant number 262067, funded by the European Union 7th Framework Program. Additionally, The Netherlands Proteomics Centre, a program embedded in The Netherlands Genomics Initiative, is kindly acknowledged for financial support as well as The Netherlands Organization for Scientific Research (NWO) with the VIDI grant (700.10.429). Work in the Mechtler lab was supported by the European Commission via the FP7 projects MeioSys and PRIME-XS, the Austrian Science Fund via the Special Research Program Chromosome Dynamics (SFB-F3402).

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    Tyrosine kinases play a prominent role in human cancer, yet the oncogenic signaling pathways driving cell proliferation and survival have been difficult to identify, in part because of the complexity of the pathways and in part because of low cellular levels of tyrosine phosphorylation. In general, global phosphoproteomic approaches reveal small nos. of peptides contg. phosphotyrosine. We have developed a strategy that emphasizes the phosphotyrosine component of the phosphoproteome and identifies large nos. of tyrosine phosphorylation sites. Peptides contg. phosphotyrosine are isolated directly from protease-digested cellular protein exts. with a phosphotyrosine-specific antibody and are identified by tandem mass spectrometry. Applying this approach to several cell systems, including cancer cell lines, shows it can be used to identify activated protein kinases and their phosphorylated substrates without prior knowledge of the signaling networks that are activated, a first step in profiling normal and oncogenic signaling networks.
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  8. 8
    Ficarro, S. B.; McCleland, M. L.; Stukenberg, P. T.; Burke, D. J.; Ross, M. M.; Shabanowitz, J.; Hunt, D. F.; White, F. M. Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae Nat. Biotechnol. 2002, 20 (3) 301– 5
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  9. 9
    Zhou, H.; Low, T. Y.; Hennrich, M. L.; van der Toorn, H.; Schwend, T.; Zou, H.; Mohammed, S.; Heck, A. J. Enhancing the identification of phosphopeptides from putative basophilic kinase substrates using Ti (IV) based IMAC enrichment Mol. Cell. Proteomics 2011, 10 (10) M110 006452
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    Boersema, P. J.; Mohammed, S.; Heck, A. J. Phosphopeptide fragmentation and analysis by mass spectrometry J. Mass Spectrom. 2009, 44 (6) 861– 78
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    10
    Phosphopeptide fragmentation and analysis by mass spectrometry
    Boersema, Paul J.; Mohammed, Shabaz; Heck, Albert J. R.
    Journal of Mass Spectrometry (2009), 44 (6), 861-878CODEN: JMSPFJ; ISSN:1076-5174. (John Wiley & Sons Ltd.)
    A review. Reversible phosphorylation is a key event in many biol. processes and is therefore a much studied phenomenon. The mass spectrometric (MS) anal. of phosphorylation is challenged by the substoichiometric levels of phosphorylation and the lability of the phosphate group in collision-induced dissocn. (CID). Here, we review the fragmentation behavior of phosphorylated peptides in MS and discuss several MS approaches that have been developed to improve and facilitate the anal. of phosphorylated peptides. CID of phosphopeptides typically results in spectra dominated by a neutral loss of the phosphate group. Several proposed mechanisms for this neutral loss and several factors affecting the extent at which this occurs are discussed. Approaches are described to interpret such neutral loss-dominated spectra to identify the phosphopeptide and localize the phosphorylation site. Methods using addnl. activation, such as MS3 and multistage activation (MSA), have been designed to generate more sequence-informative fragments from the ion produced by the neutral loss. The characteristics and benefits of these methods are reviewed together with approaches using phosphopeptide derivatization or specific MS scan modes. Addnl., electron-driven dissocn. methods by electron capture dissocn. (ECD) or electron transfer dissocn. (ETD) and their application in phosphopeptide anal. are evaluated. Finally, these techniques are put into perspective for their use in large-scale phosphoproteomics studies.
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  11. 11
    Beausoleil, S. A.; Jedrychowski, M.; Schwartz, D.; Elias, J. E.; Villen, J.; Li, J.; Cohn, M. A.; Cantley, L. C.; Gygi, S. P. Large-scale characterization of HeLa cell nuclear phosphoproteins Proc. Natl. Acad. Sci. U.S.A. 2004, 101 (33) 12130– 5
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    11
    Large-scale characterization of HeLa cell nuclear phosphoproteins
    Beausoleil, Sean A.; Jedrychowski, Mark; Schwartz, Daniel; Elias, Joshua E.; Villen, Judit; Li, Jiaxu; Cohn, Martin A.; Cantley, Lewis C.; Gygi, Steven P.
    Proceedings of the National Academy of Sciences of the United States of America (2004), 101 (33), 12130-12135CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Detg. the site of a regulatory phosphorylation event is often essential for elucidating specific kinase-substrate relationships, providing a handle for understanding essential signaling pathways and ultimately allowing insights into numerous disease pathologies. Despite intense research efforts to elucidate mechanisms of protein phosphorylation regulation, efficient, large-scale identification and characterization of phosphorylation sites remains an unsolved problem. In this report we describe an application of existing technol. for the isolation and identification of phosphorylation sites. By using a strategy based on strong cation exchange chromatog., phosphopeptides were enriched from the nuclear fraction of HeLa cell lysate. From 967 proteins, 2,002 phosphorylation sites were detd. by tandem MS. This unprecedented large collection of sites permitted a detailed accounting of known and unknown kinase motifs and substrates.
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  12. 12
    Schroeder, M. J.; Shabanowitz, J.; Schwartz, J. C.; Hunt, D. F.; Coon, J. J. A neutral loss activation method for improved phosphopeptide sequence analysis by quadrupole ion trap mass spectrometry Anal. Chem. 2004, 76 (13) 3590– 8
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    12
    A neutral loss activation method for improved phosphopeptide sequence analysis by quadrupole ion trap mass spectrometry
    Schroeder, Melanie J.; Shabanowitz, Jeffrey; Schwartz, Jae C.; Hunt, Donald F.; Coon, Joshua J.
    Analytical Chemistry (2004), 76 (13), 3590-3598CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    Recent advances in phosphopeptide enrichment prior to mass spectrometric anal. show genuine promise for characterization of phosphoproteomes. Tandem mass spectrometry of phosphopeptide ions, using collision-activated dissocn. (CAD), often produces product ions dominated by the neutral loss of phosphoric acid. Here we describe a novel method, termed Pseudo MSn, for phosphopeptide ion dissocn. in quadrupole ion trap mass spectrometers. The method induces collisional activation of product ions, those resulting from neutral loss(es) of phosphoric acid, following activation of the precursor ion. Thus, the principal neutral loss product ions are converted into a variety of structurally informative species. Since product ions from both the original precursor activation and all subsequent neutral loss product activations are simultaneously stored, the method generates a "composite" spectrum contg. fragments derived from multiple precursors. In comparison to anal. by conventional MS/MS (CAD), Pseudo MSn shows improved phosphopeptide ion dissocn. for 7 out of 10 synthetic phosphopeptides, as judged by an automated search algorithm (TurboSEQUEST). A similar overall improvement was obsd. upon application of Pseudo MSn to peptides generated by enzymic digestion of a single phosphoprotein. Finally, when applied to a complex phosphopeptide mixt., several phosphopeptides misassigned by TurboSEQUEST under the conventional CAD approach were successfully identified after anal. by Pseudo MSn.
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  13. 13
    Olsen, J. V.; Macek, B.; Lange, O.; Makarov, A.; Horning, S.; Mann, M. Higher-energy C-trap dissociation for peptide modification analysis Nat. Methods 2007, 4 (9) 709– 12
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    13
    Higher-energy C-trap dissociation for peptide modification analysis
    Olsen, Jesper V.; Macek, Boris; Lange, Oliver; Makarov, Alexander; Horning, Stevan; Mann, Matthias
    Nature Methods (2007), 4 (9), 709-712CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)
    Peptide sequencing is the basis of mass spectrometry-driven proteomics. Here we show that in the linear ion trap-orbitrap mass spectrometer (LTQ Orbitrap) peptide ions can be efficiently fragmented by high-accuracy and full-mass-range tandem mass spectrometry (MS/MS) via higher-energy C-trap dissocn. (HCD). Immonium ions generated via HCD pinpoint modifications such as phosphotyrosine with very high confidence. Addnl. we show that an added octopole collision cell facilitates de novo sequencing.
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  14. 14
    Chi, A.; Huttenhower, C.; Geer, L. Y.; Coon, J. J.; Syka, J. E.; Bai, D. L.; Shabanowitz, J.; Burke, D. J.; Troyanskaya, O. G.; Hunt, D. F. Analysis of phosphorylation sites on proteins from Saccharomyces cerevisiae by electron transfer dissociation (ETD) mass spectrometry Proc. Natl. Acad. Sci. U.S.A. 2007, 104 (7) 2193– 8
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  15. 15
    Molina, H.; Matthiesen, R.; Kandasamy, K.; Pandey, A. Comprehensive comparison of collision induced dissociation and electron transfer dissociation Anal. Chem. 2008, 80 (13) 4825– 35
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    Beausoleil, S. A.; Villen, J.; Gerber, S. A.; Rush, J.; Gygi, S. P. A probability-based approach for high-throughput protein phosphorylation analysis and site localization Nat. Biotechnol. 2006, 24 (10) 1285– 92
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    16
    A probability-based approach for high-throughput protein phosphorylation analysis and site localization
    Beausoleil, Sean A.; Villen, Judit; Gerber, Scott A.; Rush, John; Gygi, Steven P.
    Nature Biotechnology (2006), 24 (10), 1285-1292CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)
    Data anal. and interpretation remain major logistical challenges when attempting to identify large nos. of protein phosphorylation sites by nanoscale reverse-phase liq. chromatog./tandem mass spectrometry (LC-MS/MS). In this report the authors address challenges that are often only addressable by laborious manual validation, including data set error, data set sensitivity and phosphorylation site localization. The authors provide a large-scale phosphorylation data set with a measured error rate as detd. by the target-decoy approach, the authors demonstrate an approach to maximize data set sensitivity by efficiently distracting incorrect peptide spectral matches (PSMs), and the authors present a probability-based score, the Ascore, that measures the probability of correct phosphorylation site localization based on the presence and intensity of site-detg. ions in MS/MS spectra. The authors applied their methods in a fully automated fashion to nocodazole-arrested HeLa cell lysate where the authors identified 1,761 nonredundant phosphorylation sites from 491 proteins with a peptide false-pos. rate of 1.3%.
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  17. 17
    Stensballe, A.; Jensen, O. N.; Olsen, J. V.; Haselmann, K. F.; Zubarev, R. A. Electron capture dissociation of singly and multiply phosphorylated peptides Rapid Commun. Mass Spectrom. 2000, 14 (19) 1793– 800
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    17
    Electron capture dissociation of singly and multiply phosphorylated peptides
    Stensballe, Allan; Jensen, Ole Norregaard; Olsen, Jesper V.; Haselmann, Kim F.; Zubarev, Roman A.
    Rapid Communications in Mass Spectrometry (2000), 14 (19), 1793-1800CODEN: RCMSEF; ISSN:0951-4198. (John Wiley & Sons Ltd.)
    Anal. of phosphotyrosine and phosphoserine contg. peptides by nano-electrospray Fourier transform ion cyclotron resonance (FTICR) mass spectrometry established electron capture dissocn. (ECD) as a viable method for phosphopeptide sequencing. In general, ECD spectra of synthetic and native phosphopeptides appeared less complex than conventional collision activated dissocn. (CAD) mass spectra of these species. ECD of multiply protonated phosphopeptide ions generated mainly c- and z·-type peptide fragment ion series. No loss of water, phosphate groups or phosphoric acid from intact phosphopeptide ions nor from the c and z· fragment ion products was obsd. in the ECD spectra. ECD enabled complete or near-complete amino acid sequencing of phosphopeptides for the assignment of up to four phosphorylation sites in peptides in the mass range 1400 to 3500 Da. Nano-scale Fe(III)-affinity chromatog. combined with nano-electrospray FTMS/ECD facilitated phosphopeptide anal. and amino acid sequencing from crude proteolytic peptide mixts.
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  18. 18
    Kelstrup, C. D.; Hekmat, O.; Francavilla, C.; Olsen, J. V. Pinpointing phosphorylation sites: Quantitative filtering and a novel site-specific x-ion fragment J. Proteome Res. 2011, 10 (7) 2937– 48
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    18
    Pinpointing phosphorylation sites: Quantitative filtering and novel site-specific x-ion fragment
    Kelstrup, Christian D.; Hekmat, Omid; Francavilla, Chiara; Olsen, Jesper V.
    Journal of Proteome Research (2011), 10 (7), 2937-2948CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)
    Phosphoproteomics deals with the identification and quantification of thousands of phosphopeptides. Localizing the phosphorylation site is however much more difficult than establishing the identity of a phosphorylated peptide. Further, recent findings have raised doubts of the validity of the site assignments in large-scale phosphoproteomics data sets. To improve methods for site localization, we made use of a synthetic phosphopeptide library and SILAC-labeled peptides from whole cell lysates and analyzed these with high-resoln. tandem mass spectrometry on an LTQ Orbitrap Velos. We validated gas-phase phosphate rearrangement reactions during collision-induced dissocn. (CID) and used these spectra to devise a quant. filter that by comparing signal intensities of putative phosphorylated fragment ions with their nonphosphorylated counterparts allowed us to accurately pinpoint which fragment ions contain a phosphorylated residue and which ones do not. We also evaluated higher-energy collisional dissocn. (HCD) and found this to be an accurate method for correct phosphorylation site localization with no gas-phase rearrangements obsd. above noise level. Analyzing a large set of HCD spectra of SILAC-labeled phosphopeptides, we identified a novel fragmentation mechanism that generates a phosphorylation site-specific neutral loss derived x-ion, which directly pinpoints the phosphorylated residue. Together, these findings significantly improve phosphorylation site localization confidence.
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  19. 19
    Shin, Y. S.; Moon, J. H.; Kim, M. S. Observation of phosphorylation site-specific dissociation of singly protonated phosphopeptides J. Am. Soc. Mass Spectrom. 2010, 21 (1) 53– 9
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    There is no corresponding record for this reference.
  20. 20
    Gehrig, P. M.; Roschitzki, B.; Rutishauser, D.; Reiland, S.; Schlapbach, R. Phosphorylated serine and threonine residues promote site-specific fragmentation of singly charged, arginine-containing peptide ions Rapid Commun. Mass Spectrom. 2009, 23 (10) 1435– 45
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    20
    Phosphorylated serine and threonine residues promote site-specific fragmentation of singly charged, arginine-containing peptide ions
    Gehrig, Peter Max; Roschitzki, Bernd; Rutishauser, Dorothea; Reiland, Sonja; Schlapbach, Ralph
    Rapid Communications in Mass Spectrometry (2009), 23 (10), 1435-1445CODEN: RCMSEF; ISSN:0951-4198. (John Wiley & Sons Ltd.)
    To investigate gas-phase fragmentation reactions of phosphorylated peptide ions, matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) tandem mass (MS/MS) spectra were recorded from synthetic phosphopeptides and from phosphopeptides isolated from natural sources. MALDI-TOF/TOF (TOF: time-of-flight) spectra of synthetic arginine-contg. phosphopeptides revealed a significant increase of y ions resulting from bond cleavages on the C-terminal side of phosphothreonine or phosphoserine. The same effect was found in ESI-MS/MS spectra recorded from the singly charged but not from the doubly charged ions of these phosphopeptides. ESI-MS/MS spectra of doubly charged phosphopeptides contg. two arginine residues support the following general fragmentation rule: Increased amide bond cleavage on the C-terminal side of phosphorylated serines or threonines mainly occurs in peptide ions which do not contain mobile protons. In MALDI-TOF/TOF spectra of phosphopeptides displaying N-terminal fragment ions, abundant b-H3PO4 ions resulting from the enhanced dissocn. of the pSer/pThr-X bond were detected (X denotes amino acids). Cleavages at phosphoamino acids were particularly predominant in spectra of phosphopeptides contg. pSer/pThr-Pro bonds. A quant. evaluation of a larger set of MALDI-TOF/TOF spectra recorded from phosphopeptides indicated that phosphoserine residues in arginine-contg. peptides increase the signal intensities of the resp. y ions by almost a factor of 3. A less pronounced cleavage-enhancing effect was obsd. in some lysine-contg. phosphopeptides without arginine. The proposed peptide fragmentation pathways involve a nucleophilic attack by phosphate oxygen on the carbon center of the peptide backbone amide, which eventually leads to cleavage of the amide bond. Copyright © 2009 John Wiley & Sons, Ltd.
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  21. 21
    Lu, B.; Ruse, C.; Xu, T.; Park, S. K.; Yates, J. R., 3rd Automatic validation of phosphopeptide identifications from tandem mass spectra Anal. Chem. 2007, 79 (4) 1301– 10
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  22. 22
    Bailey, C. M.; Sweet, S. M.; Cunningham, D. L.; Zeller, M.; Heath, J. K.; Cooper, H. J. SLoMo: automated site localization of modifications from ETD/ECD mass spectra J. Proteome Res. 2009, 8 (4) 1965– 71
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    22
    SLoMo: Automated Site Localization of Modifications from ETD/ECD Mass Spectra
    Bailey, Christopher M.; Sweet, Steve M. M.; Cunningham, Debbie L.; Zeller, Martin; Heath, John K.; Cooper, Helen J.
    Journal of Proteome Research (2009), 8 (4), 1965-1971CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)
    Recently, software has become available to automate localization of phosphorylation sites from CID (collision-induced dissocn.) data and to assign assocd. confidence scores. The authors present an algorithm, SLoMo (Site Localization of Modifications), which extends this capability to electron transfer dissocn./electron capture dissocn. (ETD/ECD) mass spectra. Furthermore, SLoMo caters for both high and low resoln. data and allows for site-localization of any UniMod post-translational modification. SLoMo accepts input data from a variety of formats (e.g., Sequest, OMSSA). The authors validate SLoMo with high and low resoln. ETD, ECD, and CID data.
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  23. 23
    Savitski, M. M.; Mathieson, T.; Becher, I.; Bantscheff, M. H-score, a mass accuracy driven rescoring approach for improved peptide identification in modification rich samples J. Proteome Res. 2010, 9 (11) 5511– 6
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    23
    H-Score, score, a mass accuracy driven rescoring approach for improved peptide identification in modification rich samples
    Savitski, Mikhail M.; Mathieson, Toby; Becher, Isabelle; Bantscheff, Marcus
    Journal of Proteome Research (2010), 9 (11), 5511-5516CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)
    Currently, scoring algorithms of many popular search engines for tandem mass spectrometry (MS/MS) data only partially utilize the information content of high mass accuracy MS/MS data. We have developed a new rescoring scheme, H-score, that employs high mass accuracy matching of all detected fragment ions to candidate peptide sequences in an abundance independent fashion. Peptides for which b or y ions are found for all or almost all backbone fragmentation sites are rewarded. For peptide hits generated by Mascot, rescoring proved to be particularly beneficial when applied on samples contg. many different potential modifications. For a histone sample acquired on an Orbitrap Velos using HCD for peptide fragmentation, the H-score identified 24% more spectra at 0.01 false pos. rate than Mascot scoring of spectra processed according to state-of-the-art methods and 61% better than Mascot scoring of unprocessed MS/MS spectra. For a low-abundance sample, where many weak spectra were detected, these nos. went up to 53 and 190%, resp. When applied on a kinase-enriched sample contg. only a few modifications, a smaller but still significant gain of 5% was obsd.
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  24. 24
    Ruttenberg, B. E.; Pisitkun, T.; Knepper, M. A.; Hoffert, J. D. PhosphoScore: an open-source phosphorylation site assignment tool for MSn data Journal of Proteome Research 2008, 7 (7) 3054– 9
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    There is no corresponding record for this reference.
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    Hansen, T. A.; Sylvester, M.; Jensen, O. N.; Kjeldsen, F. Automated and high confidence protein phosphorylation site localization using complementary collision-activated dissociation and electron transfer dissociation tandem mass spectrometry Anal. Chem. 2012, 84 (22) 9694– 9
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    Vandenbogaert, M.; Hourdel, V.; Jardin-Mathe, O.; Bigeard, J.; Bonhomme, L.; Legros, V.; Hirt, H.; Schwikowski, B.; Pflieger, D. Automated phosphopeptide identification using multiple MS/MS fragmentation modes J. Proteome Res. 2012, 11 (12) 5695– 703
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    26
    Automated Phosphopeptide Identification Using Multiple MS/MS Fragmentation Modes
    Vandenbogaert, Mathias; Hourdel, Veronique; Jardin-Mathe, Olivia; Bigeard, Jean; Bonhomme, Ludovic; Legros, Veronique; Hirt, Heribert; Schwikowski, Benno; Pflieger, Delphine
    Journal of Proteome Research (2012), 11 (12), 5695-5703CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)
    Phosphopeptide identification is still a challenging task because fragmentation spectra obtained by mass spectrometry do not necessarily contain sufficient fragment ions to establish with certainty the underlying amino acid sequence and the precise phosphosite. To improve upon this, it has been suggested to acquire pairs of spectra from every phosphorylated precursor ion using different fragmentation modes, for example CID, ETD, and/or HCD. The development of automated tools for the interpretation of these paired spectra has however, until now, lagged behind. Using phosphopeptide samples analyzed by an LTQ-Orbitrap instrument, we here assess an approach in which, on each selected precursor, a pair of CID spectra, with or without multistage activation (MSA or MS2, resp.), are acquired in the linear ion trap. We applied this approach on phosphopeptide samples of variable proteomic complexity obtained from Arabidopsis thaliana. We present a straightforward computational approach to reconcile sequence and phosphosite identifications provided by the database search engine Mascot on the spectrum pairs, using two simple filtering rules, at the amino acid sequence and phosphosite localization levels. If multiple sequences and/or phosphosites are likely, they are reported in the consensus sequence. Using our program FragMixer, we could assess that on samples of moderate complexity, it was worth combining the two fragmentation schemes on every precursor ion to help efficiently identify amino acid sequences and precisely localize phosphosites. FragMixer can be flexibly configured, independently of the Mascot search parameters, and can be applied to various spectrum pairs, such as MSA/ETD and ETD/HCD, to automatically compare and combine the information provided by these more differing fragmentation modes. The software is openly accessible and can be downloaded from our Web site at http://proteomics.fr/FragMixer.
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    Taus, T.; Kocher, T.; Pichler, P.; Paschke, C.; Schmidt, A.; Henrich, C.; Mechtler, K. Universal and confident phosphorylation site localization using phosphoRS J. Proteome Res. 2011, 10 (12) 5354– 62
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    27
    Universal and Confident Phosphorylation Site Localization Using phosphoRS
    Taus, Thomas; Koecher, Thomas; Pichler, Peter; Paschke, Carmen; Schmidt, Andreas; Henrich, Christoph; Mechtler, Karl
    Journal of Proteome Research (2011), 10 (12), 5354-5362CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)
    An algorithm for the assignment of phosphorylation sites in peptides is described. The program uses tandem mass spectrometry data in conjunction with the resp. peptide sequences to calc. site probabilities for all potential phosphorylation sites. Tandem mass spectra from synthetic phosphopeptides were used for optimization of the scoring parameters employing all commonly used fragmentation techniques. Calcn. of probabilities was adapted to the different fragmentation methods and to the max. mass deviation of the anal. The software includes a novel approach to peak extn., required for matching exptl. data to the theor. values of all isoforms, by defining individual peak depths for the different regions of the tandem mass spectrum. Mixts. of synthetic phosphopeptides were used to validate the program by calcn. of its false localization rate vs. site probability cutoff characteristic. Notably, the empirical obtained precision was higher than indicated by the applied probability cutoff. In addn., the performance of the algorithm was compared to existing approaches to site localization such as Ascore. To assess the practical applicability of the algorithm to large data sets, phosphopeptides from a biol. sample were analyzed, localizing more than 3000 nonredundant phosphorylation sites. Finally, the results obtained for the different fragmentation methods and localization tools were compared and discussed.
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  28. 28
    Frese, C. K.; Altelaar, M.; van den Toorn, H. W.; Nolting, D.; Griep-Raming, J.; Heck, A. J.; Mohammed, S. Toward full peptide sequence coverage by dual fragmentation combining electron-transfer and higher-energy collision dissociation tandem mass spectrometry Anal. Chem. 2012, 84 (22) 9668– 73
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    28
    Toward Full Peptide Sequence Coverage by Dual Fragmentation Combining Electron-Transfer and Higher-Energy Collision Dissociation Tandem Mass Spectrometry
    Frese, Christian K.; Altelaar, A. F. Maarten; van den Toorn, Henk; Nolting, Dirk; Griep-Raming, Jens; Heck, Albert J. R.; Mohammed, Shabaz
    Analytical Chemistry (Washington, DC, United States) (2012), 84 (22), 9668-9673CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
    Increasing peptide sequence coverage by tandem mass spectrometry improves confidence in database search-based peptide identification and facilitates mapping of post-translational modifications and de novo sequencing. Inducing 2-fold fragmentation by combining electron-transfer and higher-energy collision dissocn. (EThcD) generates dual fragment ion series and facilitates extensive peptide backbone fragmentation. After an initial electron-transfer dissocn. step, all ions including the unreacted precursor ions are subjected to collision induced dissocn. which yields b/y- and c/z-type fragment ions in a single spectrum. This new fragmentation scheme provides richer spectra and substantially increases the peptide sequence coverage and confidence in peptide identification.
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  29. 29
    Altelaar, A. F.; Frese, C. K.; Preisinger, C.; Hennrich, M. L.; Schram, A. W.; Timmers, H. T.; Heck, A. J.; Mohammed, S. Benchmarking stable isotope labeling based quantitative proteomics J. Proteomics 2012,  DOI: 10.1016/j.jprot.2012.10.009
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    Zhou, H.; Ye, M.; Dong, J.; Han, G.; Jiang, X.; Wu, R.; Zou, H. Specific phosphopeptide enrichment with immobilized titanium ion affinity chromatography adsorbent for phosphoproteome analysis J. Proteome Res. 2008, 7 (9) 3957– 67
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    Specific phosphopeptide enrichment with immobilized titanium ion affinity chromatography adsorbent for Phosphoproteome Analysis
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  • Abstract

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    Figure 1

    Figure 1. EThcD MS/MS spectrum of a doubly phosphorylated peptide. RGTGQSDDSDIWDDTALIK is doubly phosphorylated and contains in total four potential phosphorylation sites. EThcD generates dual ion series that enable phosphorylation site localization with very high confidence (phosphoRS site probabilities: T(3), 0.0%; S(6), 100.0%; S(9), 100.0%; T(15), 0.0%). SEQUEST Xcorr 7.79.

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    Figure 2

    Figure 2. EThcD spectrum of a proline-containing phosphopeptide. This EThcD spectrum of a doubly charged peptide that contains four serine residues, one of which is phosphorylated. ETD does not cleave the N–Cα bond N-terminal to proline and the phosphorylation site probability is only 50% based on c- and z-ions alone. Dual fragmentation by EThcD generates complementary sequence information from c/z- and b/y-ions (SEQUEST Xcorr 4.10). Here, the exact phosphosite is revealed by y-ions that cover the corresponding phosphosite (phosphoRS site probabilitis: S(1): 0.0; S(3): 0.0; S(8): 99.5; S(10): 0.5). SEQUEST Xcorr 4.10.

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      Schroeder, Melanie J.; Shabanowitz, Jeffrey; Schwartz, Jae C.; Hunt, Donald F.; Coon, Joshua J.
      Analytical Chemistry (2004), 76 (13), 3590-3598CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      Recent advances in phosphopeptide enrichment prior to mass spectrometric anal. show genuine promise for characterization of phosphoproteomes. Tandem mass spectrometry of phosphopeptide ions, using collision-activated dissocn. (CAD), often produces product ions dominated by the neutral loss of phosphoric acid. Here we describe a novel method, termed Pseudo MSn, for phosphopeptide ion dissocn. in quadrupole ion trap mass spectrometers. The method induces collisional activation of product ions, those resulting from neutral loss(es) of phosphoric acid, following activation of the precursor ion. Thus, the principal neutral loss product ions are converted into a variety of structurally informative species. Since product ions from both the original precursor activation and all subsequent neutral loss product activations are simultaneously stored, the method generates a "composite" spectrum contg. fragments derived from multiple precursors. In comparison to anal. by conventional MS/MS (CAD), Pseudo MSn shows improved phosphopeptide ion dissocn. for 7 out of 10 synthetic phosphopeptides, as judged by an automated search algorithm (TurboSEQUEST). A similar overall improvement was obsd. upon application of Pseudo MSn to peptides generated by enzymic digestion of a single phosphoprotein. Finally, when applied to a complex phosphopeptide mixt., several phosphopeptides misassigned by TurboSEQUEST under the conventional CAD approach were successfully identified after anal. by Pseudo MSn.
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    13. 13
      Olsen, J. V.; Macek, B.; Lange, O.; Makarov, A.; Horning, S.; Mann, M. Higher-energy C-trap dissociation for peptide modification analysis Nat. Methods 2007, 4 (9) 709– 12
      13
      Higher-energy C-trap dissociation for peptide modification analysis
      Olsen, Jesper V.; Macek, Boris; Lange, Oliver; Makarov, Alexander; Horning, Stevan; Mann, Matthias
      Nature Methods (2007), 4 (9), 709-712CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)
      Peptide sequencing is the basis of mass spectrometry-driven proteomics. Here we show that in the linear ion trap-orbitrap mass spectrometer (LTQ Orbitrap) peptide ions can be efficiently fragmented by high-accuracy and full-mass-range tandem mass spectrometry (MS/MS) via higher-energy C-trap dissocn. (HCD). Immonium ions generated via HCD pinpoint modifications such as phosphotyrosine with very high confidence. Addnl. we show that an added octopole collision cell facilitates de novo sequencing.
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    14. 14
      Chi, A.; Huttenhower, C.; Geer, L. Y.; Coon, J. J.; Syka, J. E.; Bai, D. L.; Shabanowitz, J.; Burke, D. J.; Troyanskaya, O. G.; Hunt, D. F. Analysis of phosphorylation sites on proteins from Saccharomyces cerevisiae by electron transfer dissociation (ETD) mass spectrometry Proc. Natl. Acad. Sci. U.S.A. 2007, 104 (7) 2193– 8
      There is no corresponding record for this reference.
    15. 15
      Molina, H.; Matthiesen, R.; Kandasamy, K.; Pandey, A. Comprehensive comparison of collision induced dissociation and electron transfer dissociation Anal. Chem. 2008, 80 (13) 4825– 35
      There is no corresponding record for this reference.
    16. 16
      Beausoleil, S. A.; Villen, J.; Gerber, S. A.; Rush, J.; Gygi, S. P. A probability-based approach for high-throughput protein phosphorylation analysis and site localization Nat. Biotechnol. 2006, 24 (10) 1285– 92
      16
      A probability-based approach for high-throughput protein phosphorylation analysis and site localization
      Beausoleil, Sean A.; Villen, Judit; Gerber, Scott A.; Rush, John; Gygi, Steven P.
      Nature Biotechnology (2006), 24 (10), 1285-1292CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)
      Data anal. and interpretation remain major logistical challenges when attempting to identify large nos. of protein phosphorylation sites by nanoscale reverse-phase liq. chromatog./tandem mass spectrometry (LC-MS/MS). In this report the authors address challenges that are often only addressable by laborious manual validation, including data set error, data set sensitivity and phosphorylation site localization. The authors provide a large-scale phosphorylation data set with a measured error rate as detd. by the target-decoy approach, the authors demonstrate an approach to maximize data set sensitivity by efficiently distracting incorrect peptide spectral matches (PSMs), and the authors present a probability-based score, the Ascore, that measures the probability of correct phosphorylation site localization based on the presence and intensity of site-detg. ions in MS/MS spectra. The authors applied their methods in a fully automated fashion to nocodazole-arrested HeLa cell lysate where the authors identified 1,761 nonredundant phosphorylation sites from 491 proteins with a peptide false-pos. rate of 1.3%.
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    17. 17
      Stensballe, A.; Jensen, O. N.; Olsen, J. V.; Haselmann, K. F.; Zubarev, R. A. Electron capture dissociation of singly and multiply phosphorylated peptides Rapid Commun. Mass Spectrom. 2000, 14 (19) 1793– 800
      17
      Electron capture dissociation of singly and multiply phosphorylated peptides
      Stensballe, Allan; Jensen, Ole Norregaard; Olsen, Jesper V.; Haselmann, Kim F.; Zubarev, Roman A.
      Rapid Communications in Mass Spectrometry (2000), 14 (19), 1793-1800CODEN: RCMSEF; ISSN:0951-4198. (John Wiley & Sons Ltd.)
      Anal. of phosphotyrosine and phosphoserine contg. peptides by nano-electrospray Fourier transform ion cyclotron resonance (FTICR) mass spectrometry established electron capture dissocn. (ECD) as a viable method for phosphopeptide sequencing. In general, ECD spectra of synthetic and native phosphopeptides appeared less complex than conventional collision activated dissocn. (CAD) mass spectra of these species. ECD of multiply protonated phosphopeptide ions generated mainly c- and z·-type peptide fragment ion series. No loss of water, phosphate groups or phosphoric acid from intact phosphopeptide ions nor from the c and z· fragment ion products was obsd. in the ECD spectra. ECD enabled complete or near-complete amino acid sequencing of phosphopeptides for the assignment of up to four phosphorylation sites in peptides in the mass range 1400 to 3500 Da. Nano-scale Fe(III)-affinity chromatog. combined with nano-electrospray FTMS/ECD facilitated phosphopeptide anal. and amino acid sequencing from crude proteolytic peptide mixts.
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    18. 18
      Kelstrup, C. D.; Hekmat, O.; Francavilla, C.; Olsen, J. V. Pinpointing phosphorylation sites: Quantitative filtering and a novel site-specific x-ion fragment J. Proteome Res. 2011, 10 (7) 2937– 48
      18
      Pinpointing phosphorylation sites: Quantitative filtering and novel site-specific x-ion fragment
      Kelstrup, Christian D.; Hekmat, Omid; Francavilla, Chiara; Olsen, Jesper V.
      Journal of Proteome Research (2011), 10 (7), 2937-2948CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)
      Phosphoproteomics deals with the identification and quantification of thousands of phosphopeptides. Localizing the phosphorylation site is however much more difficult than establishing the identity of a phosphorylated peptide. Further, recent findings have raised doubts of the validity of the site assignments in large-scale phosphoproteomics data sets. To improve methods for site localization, we made use of a synthetic phosphopeptide library and SILAC-labeled peptides from whole cell lysates and analyzed these with high-resoln. tandem mass spectrometry on an LTQ Orbitrap Velos. We validated gas-phase phosphate rearrangement reactions during collision-induced dissocn. (CID) and used these spectra to devise a quant. filter that by comparing signal intensities of putative phosphorylated fragment ions with their nonphosphorylated counterparts allowed us to accurately pinpoint which fragment ions contain a phosphorylated residue and which ones do not. We also evaluated higher-energy collisional dissocn. (HCD) and found this to be an accurate method for correct phosphorylation site localization with no gas-phase rearrangements obsd. above noise level. Analyzing a large set of HCD spectra of SILAC-labeled phosphopeptides, we identified a novel fragmentation mechanism that generates a phosphorylation site-specific neutral loss derived x-ion, which directly pinpoints the phosphorylated residue. Together, these findings significantly improve phosphorylation site localization confidence.
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    19. 19
      Shin, Y. S.; Moon, J. H.; Kim, M. S. Observation of phosphorylation site-specific dissociation of singly protonated phosphopeptides J. Am. Soc. Mass Spectrom. 2010, 21 (1) 53– 9
      There is no corresponding record for this reference.
    20. 20
      Gehrig, P. M.; Roschitzki, B.; Rutishauser, D.; Reiland, S.; Schlapbach, R. Phosphorylated serine and threonine residues promote site-specific fragmentation of singly charged, arginine-containing peptide ions Rapid Commun. Mass Spectrom. 2009, 23 (10) 1435– 45
      20
      Phosphorylated serine and threonine residues promote site-specific fragmentation of singly charged, arginine-containing peptide ions
      Gehrig, Peter Max; Roschitzki, Bernd; Rutishauser, Dorothea; Reiland, Sonja; Schlapbach, Ralph
      Rapid Communications in Mass Spectrometry (2009), 23 (10), 1435-1445CODEN: RCMSEF; ISSN:0951-4198. (John Wiley & Sons Ltd.)
      To investigate gas-phase fragmentation reactions of phosphorylated peptide ions, matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) tandem mass (MS/MS) spectra were recorded from synthetic phosphopeptides and from phosphopeptides isolated from natural sources. MALDI-TOF/TOF (TOF: time-of-flight) spectra of synthetic arginine-contg. phosphopeptides revealed a significant increase of y ions resulting from bond cleavages on the C-terminal side of phosphothreonine or phosphoserine. The same effect was found in ESI-MS/MS spectra recorded from the singly charged but not from the doubly charged ions of these phosphopeptides. ESI-MS/MS spectra of doubly charged phosphopeptides contg. two arginine residues support the following general fragmentation rule: Increased amide bond cleavage on the C-terminal side of phosphorylated serines or threonines mainly occurs in peptide ions which do not contain mobile protons. In MALDI-TOF/TOF spectra of phosphopeptides displaying N-terminal fragment ions, abundant b-H3PO4 ions resulting from the enhanced dissocn. of the pSer/pThr-X bond were detected (X denotes amino acids). Cleavages at phosphoamino acids were particularly predominant in spectra of phosphopeptides contg. pSer/pThr-Pro bonds. A quant. evaluation of a larger set of MALDI-TOF/TOF spectra recorded from phosphopeptides indicated that phosphoserine residues in arginine-contg. peptides increase the signal intensities of the resp. y ions by almost a factor of 3. A less pronounced cleavage-enhancing effect was obsd. in some lysine-contg. phosphopeptides without arginine. The proposed peptide fragmentation pathways involve a nucleophilic attack by phosphate oxygen on the carbon center of the peptide backbone amide, which eventually leads to cleavage of the amide bond. Copyright © 2009 John Wiley & Sons, Ltd.
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    21. 21
      Lu, B.; Ruse, C.; Xu, T.; Park, S. K.; Yates, J. R., 3rd Automatic validation of phosphopeptide identifications from tandem mass spectra Anal. Chem. 2007, 79 (4) 1301– 10
      There is no corresponding record for this reference.
    22. 22
      Bailey, C. M.; Sweet, S. M.; Cunningham, D. L.; Zeller, M.; Heath, J. K.; Cooper, H. J. SLoMo: automated site localization of modifications from ETD/ECD mass spectra J. Proteome Res. 2009, 8 (4) 1965– 71
      22
      SLoMo: Automated Site Localization of Modifications from ETD/ECD Mass Spectra
      Bailey, Christopher M.; Sweet, Steve M. M.; Cunningham, Debbie L.; Zeller, Martin; Heath, John K.; Cooper, Helen J.
      Journal of Proteome Research (2009), 8 (4), 1965-1971CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)
      Recently, software has become available to automate localization of phosphorylation sites from CID (collision-induced dissocn.) data and to assign assocd. confidence scores. The authors present an algorithm, SLoMo (Site Localization of Modifications), which extends this capability to electron transfer dissocn./electron capture dissocn. (ETD/ECD) mass spectra. Furthermore, SLoMo caters for both high and low resoln. data and allows for site-localization of any UniMod post-translational modification. SLoMo accepts input data from a variety of formats (e.g., Sequest, OMSSA). The authors validate SLoMo with high and low resoln. ETD, ECD, and CID data.
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    23. 23
      Savitski, M. M.; Mathieson, T.; Becher, I.; Bantscheff, M. H-score, a mass accuracy driven rescoring approach for improved peptide identification in modification rich samples J. Proteome Res. 2010, 9 (11) 5511– 6
      23
      H-Score, score, a mass accuracy driven rescoring approach for improved peptide identification in modification rich samples
      Savitski, Mikhail M.; Mathieson, Toby; Becher, Isabelle; Bantscheff, Marcus
      Journal of Proteome Research (2010), 9 (11), 5511-5516CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)
      Currently, scoring algorithms of many popular search engines for tandem mass spectrometry (MS/MS) data only partially utilize the information content of high mass accuracy MS/MS data. We have developed a new rescoring scheme, H-score, that employs high mass accuracy matching of all detected fragment ions to candidate peptide sequences in an abundance independent fashion. Peptides for which b or y ions are found for all or almost all backbone fragmentation sites are rewarded. For peptide hits generated by Mascot, rescoring proved to be particularly beneficial when applied on samples contg. many different potential modifications. For a histone sample acquired on an Orbitrap Velos using HCD for peptide fragmentation, the H-score identified 24% more spectra at 0.01 false pos. rate than Mascot scoring of spectra processed according to state-of-the-art methods and 61% better than Mascot scoring of unprocessed MS/MS spectra. For a low-abundance sample, where many weak spectra were detected, these nos. went up to 53 and 190%, resp. When applied on a kinase-enriched sample contg. only a few modifications, a smaller but still significant gain of 5% was obsd.
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    24. 24
      Ruttenberg, B. E.; Pisitkun, T.; Knepper, M. A.; Hoffert, J. D. PhosphoScore: an open-source phosphorylation site assignment tool for MSn data Journal of Proteome Research 2008, 7 (7) 3054– 9
      There is no corresponding record for this reference.
    25. 25
      Hansen, T. A.; Sylvester, M.; Jensen, O. N.; Kjeldsen, F. Automated and high confidence protein phosphorylation site localization using complementary collision-activated dissociation and electron transfer dissociation tandem mass spectrometry Anal. Chem. 2012, 84 (22) 9694– 9
      There is no corresponding record for this reference.
    26. 26
      Vandenbogaert, M.; Hourdel, V.; Jardin-Mathe, O.; Bigeard, J.; Bonhomme, L.; Legros, V.; Hirt, H.; Schwikowski, B.; Pflieger, D. Automated phosphopeptide identification using multiple MS/MS fragmentation modes J. Proteome Res. 2012, 11 (12) 5695– 703
      26
      Automated Phosphopeptide Identification Using Multiple MS/MS Fragmentation Modes
      Vandenbogaert, Mathias; Hourdel, Veronique; Jardin-Mathe, Olivia; Bigeard, Jean; Bonhomme, Ludovic; Legros, Veronique; Hirt, Heribert; Schwikowski, Benno; Pflieger, Delphine
      Journal of Proteome Research (2012), 11 (12), 5695-5703CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)
      Phosphopeptide identification is still a challenging task because fragmentation spectra obtained by mass spectrometry do not necessarily contain sufficient fragment ions to establish with certainty the underlying amino acid sequence and the precise phosphosite. To improve upon this, it has been suggested to acquire pairs of spectra from every phosphorylated precursor ion using different fragmentation modes, for example CID, ETD, and/or HCD. The development of automated tools for the interpretation of these paired spectra has however, until now, lagged behind. Using phosphopeptide samples analyzed by an LTQ-Orbitrap instrument, we here assess an approach in which, on each selected precursor, a pair of CID spectra, with or without multistage activation (MSA or MS2, resp.), are acquired in the linear ion trap. We applied this approach on phosphopeptide samples of variable proteomic complexity obtained from Arabidopsis thaliana. We present a straightforward computational approach to reconcile sequence and phosphosite identifications provided by the database search engine Mascot on the spectrum pairs, using two simple filtering rules, at the amino acid sequence and phosphosite localization levels. If multiple sequences and/or phosphosites are likely, they are reported in the consensus sequence. Using our program FragMixer, we could assess that on samples of moderate complexity, it was worth combining the two fragmentation schemes on every precursor ion to help efficiently identify amino acid sequences and precisely localize phosphosites. FragMixer can be flexibly configured, independently of the Mascot search parameters, and can be applied to various spectrum pairs, such as MSA/ETD and ETD/HCD, to automatically compare and combine the information provided by these more differing fragmentation modes. The software is openly accessible and can be downloaded from our Web site at http://proteomics.fr/FragMixer.
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    27. 27
      Taus, T.; Kocher, T.; Pichler, P.; Paschke, C.; Schmidt, A.; Henrich, C.; Mechtler, K. Universal and confident phosphorylation site localization using phosphoRS J. Proteome Res. 2011, 10 (12) 5354– 62
      27
      Universal and Confident Phosphorylation Site Localization Using phosphoRS
      Taus, Thomas; Koecher, Thomas; Pichler, Peter; Paschke, Carmen; Schmidt, Andreas; Henrich, Christoph; Mechtler, Karl
      Journal of Proteome Research (2011), 10 (12), 5354-5362CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)
      An algorithm for the assignment of phosphorylation sites in peptides is described. The program uses tandem mass spectrometry data in conjunction with the resp. peptide sequences to calc. site probabilities for all potential phosphorylation sites. Tandem mass spectra from synthetic phosphopeptides were used for optimization of the scoring parameters employing all commonly used fragmentation techniques. Calcn. of probabilities was adapted to the different fragmentation methods and to the max. mass deviation of the anal. The software includes a novel approach to peak extn., required for matching exptl. data to the theor. values of all isoforms, by defining individual peak depths for the different regions of the tandem mass spectrum. Mixts. of synthetic phosphopeptides were used to validate the program by calcn. of its false localization rate vs. site probability cutoff characteristic. Notably, the empirical obtained precision was higher than indicated by the applied probability cutoff. In addn., the performance of the algorithm was compared to existing approaches to site localization such as Ascore. To assess the practical applicability of the algorithm to large data sets, phosphopeptides from a biol. sample were analyzed, localizing more than 3000 nonredundant phosphorylation sites. Finally, the results obtained for the different fragmentation methods and localization tools were compared and discussed.
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    28. 28
      Frese, C. K.; Altelaar, M.; van den Toorn, H. W.; Nolting, D.; Griep-Raming, J.; Heck, A. J.; Mohammed, S. Toward full peptide sequence coverage by dual fragmentation combining electron-transfer and higher-energy collision dissociation tandem mass spectrometry Anal. Chem. 2012, 84 (22) 9668– 73
      28
      Toward Full Peptide Sequence Coverage by Dual Fragmentation Combining Electron-Transfer and Higher-Energy Collision Dissociation Tandem Mass Spectrometry
      Frese, Christian K.; Altelaar, A. F. Maarten; van den Toorn, Henk; Nolting, Dirk; Griep-Raming, Jens; Heck, Albert J. R.; Mohammed, Shabaz
      Analytical Chemistry (Washington, DC, United States) (2012), 84 (22), 9668-9673CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      Increasing peptide sequence coverage by tandem mass spectrometry improves confidence in database search-based peptide identification and facilitates mapping of post-translational modifications and de novo sequencing. Inducing 2-fold fragmentation by combining electron-transfer and higher-energy collision dissocn. (EThcD) generates dual fragment ion series and facilitates extensive peptide backbone fragmentation. After an initial electron-transfer dissocn. step, all ions including the unreacted precursor ions are subjected to collision induced dissocn. which yields b/y- and c/z-type fragment ions in a single spectrum. This new fragmentation scheme provides richer spectra and substantially increases the peptide sequence coverage and confidence in peptide identification.
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    29. 29
      Altelaar, A. F.; Frese, C. K.; Preisinger, C.; Hennrich, M. L.; Schram, A. W.; Timmers, H. T.; Heck, A. J.; Mohammed, S. Benchmarking stable isotope labeling based quantitative proteomics J. Proteomics 2012,  DOI: 10.1016/j.jprot.2012.10.009
      There is no corresponding record for this reference.
    30. 30
      Zhou, H.; Ye, M.; Dong, J.; Han, G.; Jiang, X.; Wu, R.; Zou, H. Specific phosphopeptide enrichment with immobilized titanium ion affinity chromatography adsorbent for phosphoproteome analysis J. Proteome Res. 2008, 7 (9) 3957– 67
      30
      Specific phosphopeptide enrichment with immobilized titanium ion affinity chromatography adsorbent for Phosphoproteome Analysis
      Zhou, Houjiang; Ye, Mingliang; Dong, Jing; Han, Guanghui; Jiang, Xinning; Wu, Renan; Zou, Hanfa
      Journal of Proteome Research (2008), 7 (9), 3957-3967CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)
      The elucidation of protein post-translational modifications, such as phosphorylation, remains a challenging anal. task for proteomic studies. Since many of the proteins targeted for phosphorylation are low in abundance and phosphorylation is typically substoichiometric, a prerequisite for their identification is the specific enrichment of phosphopeptide prior to mass spectrometric anal. Here, we presented a new method termed as immobilized titanium ion affinity chromatog. (Ti4+-IMAC) for enriching phosphopeptides. A phosphate polymer, which was prepd. by direct polymn. of monomers contg. phosphate groups, was applied to immobilize Ti4+ through the chelating interaction between phosphate groups on the polymer and Ti4+. The resulting Ti4+-IMAC resin specifically isolates phosphopeptides from a digest mixt. of std. phosphoproteins and nonphosphoprotein (BSA) in a ratio as low as 1:500. Ti4+-IMAC was further applied for phosphoproteome anal. of mouse liver. We also compared Ti4+-IMAC to other enrichment methods including Fe3+-IMAC, Zr4+-IMAC, TiO2 and ZrO2, and demonstrate superior selectivity and efficiency of Ti4+-IMAC for the isolation and enrichment of phosphopeptides. The high specificity and efficiency of phosphopeptide enrichment by Ti4+-IMAC mainly resulted from the flexibility of immobilized titanium ion with spacer arm linked to polymer beads as well as the specific interaction between immobilized titanium ion and phosphate group on phosphopeptides.
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    31. 31
      Yu, Z.; Han, G.; Sun, S.; Jiang, X.; Chen, R.; Wang, F.; Wu, R.; Ye, M.; Zou, H. Preparation of monodisperse immobilized Ti(4+) affinity chromatography microspheres for specific enrichment of phosphopeptides Anal. Chim. Acta 2009, 636 (1) 34– 41
      There is no corresponding record for this reference.
    32. 32
      Zhou, H.; Ye, M.; Dong, J.; Corradini, E.; Cristobal, A.; Heck, A. J. R.; Zou, H.; Mohammed, S. Robust phosphoproteome enrichment using monodisperse microspheres-based immobilized titanium (IV) ion affinity chromatography. Nat. Protoc. 2012, accepted
      There is no corresponding record for this reference.
    33. 33
      Kall, L.; Canterbury, J. D.; Weston, J.; Noble, W. S.; MacCoss, M. J. Semi-supervised learning for peptide identification from shotgun proteomics datasets Nat. Methods 2007, 4 (11) 923– 5
      33
      Semi-supervised learning for peptide identification from shotgun proteomics datasets
      Kall Lukas; Canterbury Jesse D; Weston Jason; Noble William Stafford; MacCoss Michael J
      Nature methods (2007), 4 (11), 923-5 ISSN:1548-7091.
      Shotgun proteomics uses liquid chromatography-tandem mass spectrometry to identify proteins in complex biological samples. We describe an algorithm, called Percolator, for improving the rate of confident peptide identifications from a collection of tandem mass spectra. Percolator uses semi-supervised machine learning to discriminate between correct and decoy spectrum identifications, correctly assigning peptides to 17% more spectra from a tryptic Saccharomyces cerevisiae dataset, and up to 77% more spectra from non-tryptic digests, relative to a fully supervised approach.
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    34. 34
      Mertins, P.; Udeshi, N. D.; Clauser, K. R.; Mani, D. R.; Patel, J.; Ong, S. E.; Jaffe, J. D.; Carr, S. A. iTRAQ labeling is superior to mTRAQ for quantitative global proteomics and phosphoproteomics Mol. Cell. Proteomics 2012, 11 (6) M111 014423
      There is no corresponding record for this reference.
    35. 35
      Kandasamy, K.; Pandey, A.; Molina, H. Evaluation of Several MS/MS Search Algorithms for Analysis of Spectra Derived from Electron Transfer Dissociation Experiments Anal. Chem. 2009, 81 (17) 7170– 80
      There is no corresponding record for this reference.
    36. 36
      Elias, J. E.; Gygi, S. P. Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry Nat. Methods 2007, 4 (3) 207– 14
      36
      Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry
      Elias, Joshua E.; Gygi, Steven P.
      Nature Methods (2007), 4 (3), 207-214CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)
      Liq. chromatog. and tandem mass spectrometry (LC-MS/MS) has become the preferred method for conducting large-scale surveys of proteomes. Automated interpretation of tandem mass spectrometry (MS/MS) spectra can be problematic, however, for a variety of reasons. As most sequence search engines return results even for 'unmatchable' spectra, proteome researchers must devise ways to distinguish correct from incorrect peptide identifications. The target-decoy search strategy represents a straightforward and effective way to manage this effort. Despite the apparent simplicity of this method, some controversy surrounds its successful application. Here the authors clarify their preferred methodol. by addressing 4 issues based on obsd. decoy hit frequencies: (i) the major assumptions made with this database search strategy are reasonable; (ii) concatenated target-decoy database searches are preferable to sep. target and decoy database searches; (iii) the theor. error assocd. with target-decoy false pos. (FP) rate measurements can be estd.; and (iv) alternate methods for constructing decoy databases are similarly effective once certain considerations are taken into account.
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    37. 37
      Horn, D. M.; Zubarev, R. A.; McLafferty, F. W. Automated de novo sequencing of proteins by tandem high-resolution mass spectrometry Proc. Natl. Acad. Sci. U.S.A. 2000, 97 (19) 10313– 7
      There is no corresponding record for this reference.
    38. 38
      Cooper, H. J.; Hudgins, R. R.; Hakansson, K.; Marshall, A. G. Secondary fragmentation of linear peptides in electron capture dissociation Int. J. Mass Spectrom. 2003, 228 (2–3) 723– 8
      There is no corresponding record for this reference.
    39. 39
      Li, W.; Song, C.; Bailey, D. J.; Tseng, G. C.; Coon, J. J.; Wysocki, V. H. Statistical analysis of electron transfer dissociation pairwise fragmentation patterns Anal. Chem. 2011, 83 (24) 9540– 5
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      Kim, M. S.; Zhong, J.; Kandasamy, K.; Delanghe, B.; Pandey, A. Systematic evaluation of alternating CID and ETD fragmentation for phosphorylated peptides Proteomics 2011, 11 (12) 2568– 72
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    41. 41
      Nielsen, M. L.; Savitski, M. M.; Zubarev, R. A. Improving protein identification using complementary fragmentation techniques in fourier transform mass spectrometry Mol. Cell. Proteomics 2005, 4 (6) 835– 45
      41
      Improving protein identification using complementary fragmentation techniques in Fourier transform mass spectrometry
      Nielsen, Michael L.; Savitski, Mikhail M.; Zubarev, Roman A.
      Molecular and Cellular Proteomics (2005), 4 (6), 835-845CODEN: MCPOBS; ISSN:1535-9476. (American Society for Biochemistry and Molecular Biology)
      Identification of proteins by MS/MS is performed by matching exptl. mass spectra against calcd. spectra of all possible peptides in a protein data base. The search engine assigns each spectrum a score indicating how well the exptl. data complies with the expected one; a higher score means increased confidence in the identification. One problem is the false-pos. identifications, which arise from incomplete data as well as from the presence of misleading ions in exptl. mass spectra due to gas-phase reactions, stray ions, contaminants, and electronic noise. The authors employed a novel technique of redn. of false positives that is based on a combined use of orthogonal fragmentation techniques electron capture dissocn. (ECD) and collisionally activated dissocn. (CAD). Since ECD and CAD exhibit many complementary properties, their combined use greatly increased the anal. specificity, which was further strengthened by the high mass accuracy (≈1 ppm) afforded by Fourier transform mass spectrometry. The utility of this approach is demonstrated on a whole cell lysate from Escherichia coli. Anal. was made using the data-dependent acquisition mode. Extn. of complementary sequence information was performed prior to data base search using inhouse written software. Only masses involved in complementary pairs in the MS/MS spectrum from the same or orthogonal fragmentation techniques were submitted to the data base search. ECD/CAD identified twice as many proteins at a fixed statistically significant confidence level with on av. a 64% higher Mascot score. The confidence in protein identification was hereby increased by more than 1 order of magnitude. The combined ECD/CAD searches were on av. 20% faster than CAD-only searches. A specially developed test with scrambled MS/MS data revealed that the amt. of false-pos. identifications was dramatically reduced by the combined use of CAD and ECD.
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