Document Detail

Strong cation exchange chromatography in analysis of posttranslational modifications: innovations and perspectives.
Jump to Full Text
MedLine Citation:
PMID:  22174558     Owner:  NLM     Status:  MEDLINE    
Strong cation exchange (SCX) chromatography has been utilized as an excellent separation technique that can be combined with reversed-phase (RP) chromatography, which is frequently used in peptide mass spectrometry. Although SCX is valuable as the second component of such two-dimensional separation methods, its application goes far beyond efficient fractionation of complex peptide mixtures. Here I describe how SCX facilitates mapping of the protein posttranslational modifications (PTMs), specifically phosphorylation and N-terminal acetylation. The SCX chromatography has been mainly used for enrichment of these two PTMs, but it might also be beneficial for high-throughput analysis of other modifications that alter the net charge of a peptide.
Mariola J Edelmann
Related Documents :
21205098 - Effect of allicin on the production of polysaccharide intercellular adhesin in staphylo...
21894918 - Mass spectrometric and theoretical study of polyiodides: the connection between solid s...
22038778 - An efficient combination of supercritical fluid extraction and high-speed counter-curre...
22035698 - Hydrodynamic chromatography of macromolecules using polymer monolithic columns.
18546218 - Enhanced cellulase production in mutants of thermomonospora curvata.
18371628 - Isotachophoretic determination of hydrosulfite and metabisulfite in technical samples.
Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't; Review     Date:  2011-11-17
Journal Detail:
Title:  Journal of biomedicine & biotechnology     Volume:  2011     ISSN:  1110-7251     ISO Abbreviation:  J. Biomed. Biotechnol.     Publication Date:  2011  
Date Detail:
Created Date:  2011-12-16     Completed Date:  2012-04-17     Revised Date:  2013-06-27    
Medline Journal Info:
Nlm Unique ID:  101135740     Medline TA:  J Biomed Biotechnol     Country:  United States    
Other Details:
Languages:  eng     Pagination:  936508     Citation Subset:  IM    
Institute for Genomics, Biocomputing & Biotechnology, Mississippi Agricultural and Forestry Experiment Station, Mississippi State University, MS 39762, USA.
Export Citation:
APA/MLA Format     Download EndNote     Download BibTex
MeSH Terms
Arginine / chemistry
Biotechnology / methods*
Chromatography / methods
Chromatography, High Pressure Liquid / methods
Chromatography, Ion Exchange / methods*
Drosophila melanogaster / metabolism
Histones / chemistry
Lysine / chemistry
Mass Spectrometry / methods
Peptides / chemistry
Polysaccharides / chemistry
Protein Processing, Post-Translational*
Protein Structure, Tertiary
Proteomics / methods
Signal Transduction
Sulfonic Acids / chemistry
Reg. No./Substance:
0/Cations; 0/Histones; 0/Peptides; 0/Polysaccharides; 0/Sulfonic Acids; 56-87-1/Lysine; 74-79-3/Arginine

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

Full Text
Journal Information
Journal ID (nlm-ta): J Biomed Biotechnol
Journal ID (publisher-id): JBB
ISSN: 1110-7243
ISSN: 1110-7251
Publisher: Hindawi Publishing Corporation
Article Information
Download PDF
Copyright © 2011 Mariola J. Edelmann.
Received Day: 10 Month: 5 Year: 2011
Accepted Day: 7 Month: 9 Year: 2011
Print publication date: Year: 2011
Electronic publication date: Day: 17 Month: 11 Year: 2011
Volume: 2011E-location ID: 936508
ID: 3228578
PubMed Id: 22174558
DOI: 10.1155/2011/936508

Strong Cation Exchange Chromatography in Analysis of Posttranslational Modifications: Innovations and Perspectives
Mariola J. EdelmannI1*
Institute for Genomics, Biocomputing & Biotechnology; Mississippi Agricultural and Forestry Experiment Station, Mississippi State University, 650 Stone Boulevard, MS 39762, USA
Correspondence: *Mariola J. Edelmann:
[other] Academic Editor: George Makhatadze

1. Introduction

Posttranslational modifications (PTMs) regulate protein function, subcellular localization, and degradation. Although detection of the modified residues is crucial for understanding of the physiological roles of proteins as well as their mechanisms and pathways, it still represents a challenge. It is especially true in case of global proteome studies, in which hundreds or thousands of modified sites are monitored at the same time, and such analyses are becoming now more required due to an increasing popularity of the systems biology approaches. There is therefore a great demand for further development of efficient tools that could be used to characterize the entire complement of modified proteins in cells. Mass-spectrometry-based proteomics offers highly sensitive tools for modification mapping (reviewed in [1, 2]), where thousands of proteins can be concurrently analyzed to construct and validate comprehensive physiological models. Nevertheless, PTM analysis usually requires preceding enrichment methods due to the high complexity of the samples and the low abundance of modified peptides. In this paper I will discuss strong cation exchange (SCX) chromatography as an effective method for enrichment of PTMs, including phosphorylation and N-terminal acetylation (summarized in Table 1).

2. Principles of SCX Separation for Proteomic Applications

The SCX stationary phase usually contains aliphatic sulfonic acid groups that are negatively charged in aqueous solution, therefore tightly binding any strongly basic analytes. To recover the analyte, the resin is then washed with a solvent neutralizing this ionic interaction. Most tryptic peptides in acidic pH are characterized by a net charge of +2 and above, and they can be therefore separated by SCX from peptides possessing a net charge of +1, such as trypsin-generated phosphopeptides, C-terminal peptides, or peptides with blocked N-termini (i.e., peptides with blocked N-terminal free amine group, for instance, by N-acetylation), as well as from peptides containing higher charges, including ones containing missed cleavages and therefore more arginine and lysine residues [3]. Trypsin is not the only enzyme used to generate peptides for proteomic analyses, and other proteolytic enzymes are also utilized, for instance, Lys-N, Lys-C, Glu-C, or elastase. Some of these enzymes can be used to obtain peptides characterized by specific, distinguishable net charges, which provide additional advantages for SCX separation of differently modified peptides [4, 5].

3. SCX in Phosphoproteome Studies

The complex system of signal transduction enables effective and targeted cell responses to external or internal stimuli, and PTMs play here a major role. Phosphorylation is one of the best characterized among these PTMs. This modification has been shown to affect approximately 30% of all the human proteins [17], although recent studies suggest that this percentage is even higher [18]. Protein phosphorylation has been associated with a wide range of cellular processes, such as proliferation, migration, differentiation, cell cycle progression, and apoptosis [19, 20]. The mass spectrometric analysis of phosphopeptides can be very challenging for several reasons. Firstly, these negatively charged peptides are not easily analyzed in a positive mode, and this method of ionization is typically used in the electrospray ionization- (ESI-) based peptide mass spectrometry. Secondly, the hydrophilic property of phosphopeptides lowers their binding to the C18-based trapping columns used for purification of the peptide sample prior to its separation by HPLC. Thirdly, due to the ion suppression, detection of phosphopeptides by a mass spectrometer is problematic, especially in the complex samples containing unmodified or other more easily detectable peptides. Some of the above problems are still questionable, as discussed in [21], but the sole complexity of the peptide samples usually calls for prior enrichment in order to specifically select for phosphopeptides/phosphoproteins. These techniques include purification by phosphospecific antibodies [22, 23] and use of immobilized metal affinity chromatography (IMAC), where negatively charged phosphopeptides bind to positively charged metal ions, such as Fe3+, Ga3+, or Zr4+ [2, 2426], as well as separation by titanium dioxide (TiO2) columns [27, 28]. Second chromatography dimension is also frequently utilized, and this might include hydrophilic interaction chromatography (HILIC) [2932], electrostatic repulsion hydrophilic interaction chromatography (ERLIC) [11], and here discussed SCX.

The SCX chromatography is the most widely used HPLC fractionation method, and it has also found applications in phosphoproteomics (Table 1). The peptides are eluted from an SCX column using a low-pH buffer, and in these conditions phosphopeptides have a net charge of 0 or +1, as opposed to a majority of other tryptic peptides characterized by +2 or higher net charges. Therefore, most of the phosphopeptides elute in the first few SCX fractions, although this depends on the type of gradient used and on a number of fractions collected [3, 3335].

Because of these features, the SCX chromatography can be employed as a stand-alone method for phosphopeptide mapping, as demonstrated by Beausoleil et al. [6], who used it without additional IMAC or TiO2 phosphopeptide enrichment. In this experiment 8 mg of the nuclear protein extract derived from HeLa cells was first separated by SDS-PAGE, then ten gel bands were digested by trypsin and the peptides were resolved by SCX, where only the first four fractions were collected for subsequent LC-MS/MS analysis by LCQ DECA XP ion trap. This simple workflow allowed for identification of 2002 phosphorylation sites from 967 proteins. Adding another dimension to this separation, such as an off-gel fractionation, which is based on the isoelectric point of peptides/proteins, might result in even larger number of identified phosphopeptides without enrichment by IMAC, TiO2, or phosphospecific antibodies.

Due to the complexity of the protein samples, SCX-based phosphopeptide separation alone is often ineffective, and the secondary phosphopeptide enrichment is necessary for identification of a larger number of phosphorylated residues [33, 36, 37]. For instance, an automated SCX-TiO2-RP-LC-MS/MS method was used to separate HeLa protein digest, where 722 nonredundant phosphorylation sites were identified, including several doubly and triply phosphorylated peptides [7]. Briefly, 500 μg of HeLa protein extract was digested, and these peptide mixtures were separated either by SCX or strong anion exchange (SAX) columns. In case of SCX, each of ten SCX fractions was additionally subjected to the phosphopeptide enrichment by TiO2. The samples were analyzed by the LTQ-Orbitrap mass spectrometer combined with C18-based HPLC. This SCX method was complementary to the on-line SAX separation, which however did not use subsequent TiO2 enrichment. In comparison with SCX, the latter method was able to separate a higher number of multiply phosphorylated peptides [7], but this could be due to the TiO2 enrichment used after the SCX chromatography which is known to be less ideal for multiply phosphorylated peptide analysis. Furthermore, Sui et al. used similar SCX separation followed by TiO2 enrichment and LC-MS/MS analysis to map the phosphoproteome in human Chang liver cell line [8]. In this experiment, 1.5 mg protein was digested with trypsin and subjected to separation by SCX chromatography into forty fractions, followed by enrichment by TiO2 and LC-MS/MS analysis by Q-TOF and LCQ mass spectrometers. This study identified 1035 phosphorylation sites on 607 phosphoproteins, including a large fraction of previously unknown phosphoproteins, and the functional analysis demonstrated that majority of these proteins were involved in liver metabolism. Finally, similar procedure has been used for the phosphoproteome analysis of a Gram-negative bacterium Escherichia coli, where 81 phosphorylation sites were identified on 79 proteins [9]. In this study, 20 mg protein was digested with Lys-C followed by tryptic digestion. The resulted peptides were split into two technical replicates and separated on SCX column into ten fractions, followed by TiO2 phosphopeptide purification and analysis by HPLC coupled to LTQ-Orbitrap mass spectrometer. A comparison of this dataset to a phosphoproteomic study of bacterium Bacillus subtilis [10] indicated that both phosphoproteomes demonstrate high conservation levels, and the authors suggested that some phosphorylation sites are conserved from Archaea to humans [9]. The Bacillus subtilis study has been done using a very similar technique to the one described for E. coli [9], but 10 mg protein was used for analysis and fifteen SCX fractions were collected, which were then subjected to the TiO2 chromatography and analysis by HPLC coupled to LTQ-Orbitrap or LTQ-FT mass spectrometry.

Although SCX is a relatively old separation method, it still performs quite well if compared with newer HPLC techniques. Chen et al. [11] evaluated four different chromatography methods in terms of their capabilities to separate phosphopeptides of HeLa cells, and these included SCX, HILIC, ERLIC chromatography with nonvolatile solvent system (ERLIC-nV), and ERLIC with volatile solvent system (ERLIC-V). Each chromatography method was performed on peptides obtained from 40 mg protein. Phosphopeptides were first enriched on IMAC, followed by SCX, HILIC, ERLIC-nV, or ERLIC-V-based separation into multiple fractions (25 fractions in case of SCX) and finally analyzed by LC-MS/MS using QSTAR ELITE mass spectrometer. As a result, each technique yielded between 4000 and 5000 phosphopeptides, including a large number of nonoverlapping phosphopeptides for each HPLC method. Together, the data from all four chromatography analyses generated 9069 unique phosphopeptides, demonstrating a complementary nature of these different chromatography methods. Interestingly, the phosphopeptide resolving power was the greatest for the SCX method (60.52% nonrepetitive phosphopeptides), followed by ERLIC-nV (50.79% nonrepetitive phosphopeptides), while the lowest performance was assigned to HILIC (50.83% nonrepetitive phosphopeptides), which also had the worst orthogonality relative to reversed-phase chromatography.

Apart from combining SCX separation with additional phosphopeptide enrichment methods, another innovation is to utilize other proteolytic enzymes for peptide generation in order to complement the data obtained from trypsin-digested samples. An example is Lys-N [4, 5] that generates phosphopeptides characterized by distinct net charges, which can be thus efficiently separated by SCX and distinguished from the N-acetylated peptide populations [4]. This feature of Lys-N has been taken advantage of in a study where a parallel use of Lys-N, trypsin, and Lys-C digestion followed by SCX and LC-MS/MS analysis provided a total of 5036 nonredundant phosphopeptides [4]. In this study only 1 mg protein was subjected to digestion with one of these proteolytic enzymes. The peptides were then desalted on C18 columns and subjected to the SCX-based fractionation. Forty thus obtained SCX fractions were analyzed by HPLC combined with LTQ-Orbitrap mass spectrometry. Lys-N increased the phosphopeptide coverage by 72% if compared with the trypsin-digested sample, whereas trypsin accounted for only 25% of the increase. Clearly, future exploration of other proteolytic enzymes can additionally benefit the global phosphoproteome studies.

In addition, the mass spectrometry methods can also be modified to fully exploit the phosphopeptide separation capabilities provided by SCX. As an example, electron transfer dissociation (ETD) has emerged as an alternative to collision-induced dissociation (CID) fragmentation method used in mass spectrometry. ETD is especially suitable for analysis of highly charged peptides and for PTMs that are labile upon CID fragmentation methods [38]. As an example, this approach has been used to characterize phosphoproteome of human embryonic stem cells [12]. In this study, peptides obtained from 10 mg protein lysate were subjected to the SCX separation into twelve fractions, followed by IMAC phosphopeptide purification and LC-MS/MS analysis by the LTQ-Orbitrap mass spectrometer. The peptides were analyzed using ETD and CID fragmentation methods, which resulted in the identification of 10,844 nonredundant phosphorylation sites. Importantly, the ETD method yielded a much larger number of unique phosphopeptides than CID (8,087 in comparison with 3,868). In this study, ETD fragmentation analysis has been also shown to more frequently localize the phosphorylation site to a specific residue, which is often problematic when using CID fragmentation [12].

Finally, my preliminary data suggests that SCX chromatography resins can be further optimized in terms of their phosphopeptide enrichment properties. I used TiO2 resin as well as cartridges containing three different SCX resins to separate phosphorylated peptides of several forms of casein, followed by LS-MS/MS analysis (please, refer to the Supplementary methods in Appendix). The SCX resins included sulfonic acid (SCX-1), propyl sulfonic acid (SCX-2), and ethylbenzene sulfonic acid (SCX-3), and their performance varied in terms of their phosphopeptide enrichment potential. In this experiment, SCX-3 has been shown to be the most efficient column among the tested resins (Figure 1). Due to the presence of an aromatic ring, SCX-1 and SCX-3 resins have nonpolar character, therefore enhancing nonpolar secondary interactions with the analyte and increasing its binding to the column. However, SCX-3 might have also other characteristics that enable for a slightly better separation of phosphopeptides. This preliminary study should be repeated using a more complex sample, but it demonstrates that there might be a bigger potential for the SCX-based separation methods if these resins are further optimized specifically for phosphorylation analysis.

4. Utilizing SCX Chromatography in Analysis of N-Terminal Acetylation

The N-terminal acetylation is a common protein modification with a relatively unclear physiological role. It affects a variety of protein functions, such as enzymatic activity, DNA-binding, and protein-protein interactions [39]. It might also represent a specific degron site, thus targeting proteins for ubiquitination and subsequent degradation [40]. The SCX-based separation of N-terminal acetylated peptides is based on the fact that these peptides lack the N-terminal free amine, which contributes to their earlier elution in the SCX chromatography [13] and enables their separation from slightly later migrating phosphopeptides (Table 1; [4, 14]).

One of the first attempts to use SCX for analysis of the N-acetylated and C-terminal peptides showed that this approach, if adequately optimized, can provide 95% specificity for this peptide class [13]. The membrane-enriched fraction from 5 × 106 human teratocarcinoma Nt2/d1 cells was digested with Lys-C, followed by tryptic digestion. The peptides were desalted with a C18 column and separated by SCX into approximately thirty fractions, which were then analyzed by the LC-LTQ-Orbitrap mass spectrometry. This workflow proved to be successful, and it allowed for identification of 116 N-terminal acetylated residues, although a relatively small amount of protein material was used.

Moreover, similarly as in case of phosphoproteome analysis, the SCX-based separation of N-terminal acetylated peptides has been combined with the ETD fragmentation method [14], yielding results complementary to the ones obtained by the CID fragmentation. Digestion with proteolytic enzymes such as Lys-N and Lys-C has also been shown to be advantageous, for instance, due to already mentioned separation of the N-terminally acetylated peptides from phosphopeptides [4, 14, 16].

Furthermore, there are additional methods suitable for enrichment of the N-terminal acetylated peptides that can be used in conjunction with SCX, and these include combined fractional diagonal chromatography (COFRADIC; summarized in [41]). For instance, SCX combined with COFRADIC and LC-MS/MS analysis by an ion trap mass spectrometer has been used to identify over 1,200 mature protein N-termini, including 861 N-terminal acetylation sites in cytoplasmic, nuclear, and membrane fractions obtained from Drosophila melanogaster cells [15].

In summary, the SCX methods used for mapping of the N-terminal acetylation sites are expected to shed more light on the abundance and function of this prevalent but not well-understood modification.

5. Conclusions

Although current efforts are focused on optimizing the SCX chromatography for analysis of phosphorylation as a key signaling modification, this HPLC method has a potential to be used in analysis of other PTMs that alter a net charge of the peptides. For example, SCX could be possibly used in glycomics, based on the assumption that many extracellular N-glycans contain sialic acid residues, thereby reducing the net charge of glycopeptides in comparison with the unmodified peptides [42]. Moreover, modifications of lysine and arginine residues, such as methylation or acetylation, also account for a shift in SCX elution time in comparison with their unmodified counterparts [43]. When compared to the whole complement of peptides, these modifications might not cause a sufficient change in elution properties in order to monitor these modifications on a global scale, but they can still be optimized to study modifications of specific proteins, such as histones that are characterized by multiple lysine and arginine PTMs. Moreover, differential elution of peptides containing modified arginine or lysine residues in comparison with peptides containing these amino acids in an unmodified form can be used as an additional validation of the proteomic results. All this further underlines the importance of the SCX chromatography in proteomic applications focused on investigation of protein modifications.


M. J. Edelmann is supported by EPS-0903787. She would like to thank Dr. Benedikt Kessler (Oxford University, UK) and Alison Cleeve (Kinesis Ltd., UK) for technical assistance with the SCX cartridges. The paper was approved for publication as a Journal Article no. J-12062 of the Mississippi Agricultural and Forestry Experiment Station, Mississippi State University.

Supplementary Methods: Phosphopeptide Enrichment and Mass Spectrometry Analysis

50 μg of casein (Sigma-Aldrich, UK) was digested with trypsin, as we previously described [4446]. SCX (here referred to as SCX-1), SCX-2, and SCX-3 cartridges (ISOLUTE SCX 200 mg/3 mL; Kinesis, UK) were washed with 5 mL wash solvent containing 0.1% formic acid and 5% acetonitrile. The digested protein samples were loaded onto SCX-1, SCX-2, or SCX-3 columns and washed twice with 5 mL wash solvent. The peptides were eluted sequentially with buffers A, B, C, and D, containing 5% acetonitrile as well as 50, 100, 200, or 500 nM ammonium acetate pH 3, respectively. All the elution procedures were performed on a vacuum manifold (Kinesis, UK) connected to a vacuum pump.

For the phosphopeptide enrichment with TiO2 pipette tip (Gl Sciences, Japan), TiO2 tips were preconditioned with 100% acetonitrile, conditioned with 0.2 M phosphate buffer pH7, and equilibrated with solvent containing 50% acetonitrile and 0.1% formic acid. The peptide sample was then loaded onto the TiO2 tips, washed six times with buffer containing 50% acetonitrile, 0.1% formic acid, and 0.1 M KCl, and eluted with 0.2 M phosphate buffer pH 7 and 0.5% aqueous ammonia.

The volume of all eluates was reduced in a vacuum centrifuge (Thermo, UK) and adjusted to appropriate volume with solvent containing 2% acetonitrile and 0.1% formic acid. The triplicates of samples (~1 μg protein) were analyzed on HPLC (Ultimate 3000, Dionex, UK) attached to a 3D high-capacity ion trap mass spectrometer (Esquire HCTplus, Bruker Daltonics, UK), exactly as we previously described [4446]. The obtained peak lists were submitted to the Mascot search engine, where phosphorylation on serine, threonine, and tyrosine was added as a variable modification in the search parameters. The peptide and fragment mass tolerances were 2.5 and 0.8, respectively. Individual MS/MS spectra for peptides with a Mascot Mowse score lower than 40 were inspected manually and only included in the statistics if a series of at least four continuous y or b ions were observed. Standard variations for triplicate samples were calculated using Excel (Microsoft).

SCX: Strong cation exchange
RP: Reversed phase
PTM: Postranslational modification
ESI: Electrospray ionization
IMAC: Immobilized metal affinity chromatography
ETD: Electron transfer dissociation
CID: Collision-induced dissociation
HPLC: High-performance liquid chromatography
COFRADIC: Combined fractional diagonal chromatography
HILIC: Hydrophilic interaction chromatography
ERLIC: Electrostatic repulsion hydrophilic interaction chromatography
LC-MS/MS: Liquid chromatography-tandem mass spectrometry.

1. Mann M,Ong SE,Grønborg M,Steen H,Jensen ON,Pandey A. Analysis of protein phosphorylation using mass spectrometry: deciphering the phosphoproteomeTrends in BiotechnologyYear: 200220626126812007495
2. Wu HY,Liao PC. Analysis of protein phosphorylation using mass spectrometryChang Gung Medical JournalYear: 200831321722718782944
3. Mohammed S,Heck AJ. Strong cation exchange (SCX) based analytical methods for the targeted analysis of protein post-translational modificationsCurrent Opinion in BiotechnologyYear: 2010
4. Gauci S,Helbig AO,Slijper M,Krijgsveld J,Heck AJR,Mohammed S. Lys-N and trypsin cover complementary parts of the phosphoproteome in a refined SCX-based approachAnalytical ChemistryYear: 200981114493450119413330
5. Taouatas N,Drugan MM,Heck AJR,Mohammed S. Straightforward ladder sequencing of peptides using a Lys-N metalloendopeptidaseNature MethodsYear: 20085540540718425140
6. Beausoleil SA,Jedrychowski M,Schwartz D,et al. Large-scale characterization of HeLa cell nuclear phosphoproteinsProceedings of the National Academy of Sciences of the United States of AmericaYear: 200410133121301213515302935
7. Dai J,Wang LS,Wu YB,et al. Fully automatic separation and identification of phosphopeptides by continuous pH-gradient anion exchange online coupled with reversed-phase liquid chromatography mass spectrometryJournal of Proteome ResearchYear: 20098113314119053533
8. Sui S,Wang J,Yang B,et al. Phosphoproteome analysis of the human Chang liver cells using SCX and a complementary mass spectrometric strategyProteomicsYear: 20088102024203418491316
9. Macek B,Gnad F,Soufi B,et al. Phosphoproteome analysis of E. coli reveals evolutionary conservation of bacterial Ser/Thr/Tyr phosphorylationMolecular and Cellular ProteomicsYear: 20087229930717938405
10. Macek B,Mijakovic I,Olsen JV,et al. The serine/threonine/tyrosine phosphoproteome of the model bacterium Bacillus subtilisMolecular and Cellular ProteomicsYear: 20076469770717218307
11. Chen X,Wu D,Zhao Y,Wong BH,Guo L. Increasing phosphoproteome coverage and identification of phosphorylation motifs through combination of different HPLC fractionation methodsJournal of Chromatography BYear: 201187912534
12. Swaney DL,Wenger CD,Thomson JA,Coon JJ. Human embryonic stem cell phosphoproteome revealed by electron transfer dissociation tandem mass spectrometryProceedings of the National Academy of Sciences of the United States of AmericaYear: 20091064995100019144917
13. Dormeyer W,Mohammed S,Van Breukelen B,Krijgsveld J,Heck AJR. Targeted analysis of protein terminiJournal of Proteome ResearchYear: 20076124634464517927228
14. Taouatas N,Altelaar AFM,Drugan MM,Helbig AO,Mohammed S,Heck AJR. Strong cation exchange-based fractionation of Lys-N-generated peptides facilitates the targeted analysis of post-translational modificationsMolecular and Cellular ProteomicsYear: 20098119020018824475
15. Goetze S,Qeli E,Mosimann C,et al. Identification and functional characterization of N-terminally acetylated proteins in Drosophila melanogasterPLoS BiologyYear: 2009711 Article ID e1000236..
16. Helbig AO,Gauci S,Raijmakers R,et al. Profiling of N-acetylated protein termini provides in-depth insights into the N-terminal nature of the proteomeMolecular and Cellular ProteomicsYear: 20109592893920061308
17. Cohen P. The role of protein phosphorylation in human health and disease. The Sir Hans Krebs Medal LectureEuropean Journal of BiochemistryYear: 20012685001501011589691
18. Olsen JV,Blagoev B,Gnad F,et al. Global, in vivo, and site-specific phosphorylation dynamics in signaling networksCellYear: 2006127363564817081983
19. Manning G,Whyte DB,Martinez R,Hunter T,Sudarsanam S. The protein kinase complement of the human genomeScienceYear: 200229856001912193412471243
20. Thomas SM,Brugge JS. Cellular functions regulated by SRC family kinasesAnnual Review of Cell and Developmental BiologyYear: 199713513609
21. Steen H,Jebanathirajah JA,Rush J,Morrice N,Kirschner MW. Phosphorylation analysis by mass spectrometry: myths, facts, and the consequences for qualitative and quantitative measurementsMolecular and Cellular ProteomicsYear: 20065117218116204703
22. Pandey A,Podtelejnikov AV,Blagoev B,Bustelo XR,Mann M,Lodish HF. Analysis of receptor signaling pathways by mass spectrometry: identification of Vav-2 as a substrate of the epidermal and platelet-derived growth factor receptorsProceedings of the National Academy of Sciences of the United States of AmericaYear: 200097117918410618391
23. Grønborg M,Kristiansen TZ,Stensballe A,et al. A mass spectrometry-based proteomic approach for identification of serine/threonine-phosphorylated proteins by enrichment with phospho-specific antibodies: identification of a novel protein, Frigg, as a protein kinase A substrateMolecular and Cellular ProteomicsYear: 20021751752712239280
24. Cao P,Stults JT. Mapping the phosphorylation sites of proteins using on-line immobilized metal affinity chromatography/capillary electrophoresis/electrospray ionization multiple stage tandem mass spectrometryRapid Communications in Mass SpectrometryYear: 200014171600160610960913
25. Ficarro SB,McCleland ML,Stukenberg PT,et al. Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiaeNature BiotechnologyYear: 2002203301305
26. Nühse TS,Stensballe A,Jensen ON,Peck SC. Large-scale analysis of in vivo phosphorylated membrane proteins by immobilized metal ion affinity chromatography and mass spectrometryMolecular and Cellular ProteomicsYear: 20032111234124314506206
27. Pinkse MWH,Uitto PM,Hilhorst MJ,Ooms B,Heck AJR. Selective isolation at the femtomole level of phosphopeptides from proteolytic digests using 2D-NanoLC-ESI-MS/MS and titanium oxide precolumnsAnalytical ChemistryYear: 200476143935394315253627
28. Larsen MR,Thingholm TE,Jensen ON,Roepstorff P,Jørgensen TJD. Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumnsMolecular and Cellular ProteomicsYear: 20054787388615858219
29. Lindner H,Sarg B,Helliger W. Application of hydrophilic-interaction liquid chromatography to the separation of phosphorylated H1 histonesJournal of Chromatography AYear: 1997782155629440922
30. McNulty DE,Annan RS. Hydrophilic interaction chromatography reduces the complexity of the phosphoproteome and improves global phosphopeptide isolation and detectionMolecular and Cellular ProteomicsYear: 20087597198018212344
31. McNulty DE,Annan RS. Hydrophilic interaction chromatography for fractionation and enrichment of the phosphoproteomeMethods in Molecular BiologyYear: 20095279310519241008
32. Saleem RA,Aitchison JD. Quantitative phosphoproteomics in fatty acid stimulated Saccharomyces cerevisiaeJournal of Visualized ExperimentsYear: 200932
33. Villén J,Gygi SP. The SCX/IMAC enrichment approach for global phosphorylation analysis by mass spectrometryNature ProtocolsYear: 200831016301638
34. Tanasijevic MJ,Myers MG,Thoma RS,Crimmins DL,White MF,Sacks DB. Phosphorylation of the insulin receptor substrate IRS-1 by casein kinase IIJournal of Biological ChemistryYear: 19932682418157181668349691
35. Trinidad JC,Specht CG,Thalhammer A,Schoepfer R,Burlingame AL. Comprehensive identification of phosphorylation sites in postsynaptic density preparationsMolecular and Cellular ProteomicsYear: 20065591492216452087
36. Højlund K,Bowen BP,Hwang H,et al. In vivo phosphoproteome of human skeletal muscle revealed by phosphopeptide enrichment and HPLC-ESI-MS/MSJournal of Proteome ResearchYear: 20098114954496519764811
37. Soung GY,Miller JL,Koc H,Koc EC. Comprehensive analysis of phosphorylated proteins of Escherichia coli ribosomesJournal of Proteome ResearchYear: 2009873390340219469554
38. Kelleher NL,Zubarev RA,Bush K,et al. Localization of labile posttranslational modifications by electron capture dissociation: the case of γ-carboxyglutamic acidAnalytical ChemistryYear: 199971194250425310517147
39. Polevoda B,Sherman F. The diversity of acetylated proteinsGenome BiologyYear: 200235, article no. 00066.16.6
40. Hwang CS,Shemorry A,Varshavsky A. N-terminal acetylation of cellular proteins creates specific degradation signalsScienceYear: 2010327596897397720110468
41. Mischerikow N,Heck AJ. Targeted large-scale analysis of protein acetylationProteomicsYear: 201111457158921246731
42. Lewandrowski U,Zahedi RP,Moebius J,Walter U,Sickmann A. Enhanced N-glycosylation site analysis of sialoglycopeptides by strong cation exchange prefractionation applied to platelet plasma membranesMolecular and Cellular ProteomicsYear: 20076111933194117660510
43. Wilmarth PA,Tanner S,Dasari S,et al. Age-related changes in human crystalline determined from comparative analysis of post-translational modifications in young and aged lens: does deamidation contribute to crystallin insolubility?Journal of Proteome ResearchYear: 20065102554256617022627
44. Batycka M,Inglis NF,Cook K,et al. Ultra-fast tandem mass spectrometry scanning combined with monolithic column liquid chromatography increases throughput in proteomic analysisRapid Communications in Mass SpectrometryYear: 200620142074208016773668
45. Edelmann MJ,Kramer HB,Altun M,Kessler BM. Post-translational modification of the deubiquitinating enzyme otubain 1 modulates active RhoA levels and susceptibility to Yersinia invasionFEBS JournalYear: 2010277112515253020553488
46. Edelmann MJ,Iphöfer A,Akutsu M,et al. Structural basis and specificity of human otubain 1-mediated deubiquitinationBiochemical JournalYear: 2009418237939018954305

Article Categories:
  • Review Article

Previous Document:  CNPase expression in olfactory ensheathing cells.
Next Document:  Cytotoxic CD4 T cells in antiviral immunity.