ARTICLE pubs.acs.org/jpr
Improved Two-Dimensional Reversed Phase-Reversed Phase LC-MS/MS Approach for Identification of Peptide-Protein Interactions Heike Stephanowitz,† Sabine Lange,†,§ Diana Lang,† Christian Freund,†,‡ and Eberhard Krause*,† † ‡
Leibniz-Institut f€ur Molekulare Pharmakologie, Robert-R€ossle-Str. 10, 13125 Berlin, Germany Freie Universit€at Berlin, 14195 Berlin, Germany
bS Supporting Information ABSTRACT: Quantitative mass spectrometry (MS) in combination with affinity purification approaches allows for an unbiased study of protein-protein and peptide-protein interactions. In shotgun approaches that are based on proteolytic digestion of complex protein mixtures followed by two-dimensional liquid-phase chromatography, the separation effort prior to MS analysis is focused on tryptic peptides. Here we developed an improved offline 2-D liquid chromatographyMS/MS approach for the identification and quantification of binding proteins utilizing reversed-phase capillary columns with acidic acetonitrile-containing eluents in both chromatographic dimensions. A specific fractionation scheme was applied in order to obtain samples with evenly distributed peptides and to fully utilize the separation space in the second dimension nanoLC-MS/MS. We report peptide-protein interaction studies to identify phosphorylation-dependent binding partners of the T cell adapter protein ADAP. The results of the SILAC-based pull-down experiments show this approach is well suited for distinguishing phosphorylation-specific interactions from unspecific binding events. The data provide further evidence that phosphorylated Tyr 595 of ADAP may serve as a direct binding site for the SH2 domains of the T cell proteins SLP76 and NCK. From a technical point of view we provide a detailed protocol for an offline 2-D RPRP LC-MS/MS method that offers a robust and time-saving alternative for quantitative interactome analysis. KEYWORDS: two-dimensional liquid chromatography, quantitative proteomics, mass spectrometry, phosphorylation, protein interactions, SILAC
’ INTRODUCTION Efficient strategies for peptide identification and quantification by mass spectrometry (MS) and miniaturized peptide/ protein separation techniques are prerequisites for performing advanced proteomic studies such as interactome analysis or global quantitative protein expression profiling of subcellular structures or even whole cell lysates.1,2 A common proteomic approach that is sensitive, robust, and reproducible involves SDSPAGE separation on the protein level in combination with nanocapillary liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) analysis of peptides generated by in-gel digestion with trypsin (GeLC-MS/MS).3,4 The 1-D gel-based approach allows the identification and relative quantification of proteins over a wide range of concentration and complexity using label-free quantification,5 stable isotope labeling techniques such as SILAC6,7 and iTRAQ,8,9 or enzyme-catalyzed 18O-labeling.10,11 Alternatively, shotgun approaches, including the MudPIT methodology,12 which is based on proteolytic digestion of complex protein mixtures or even entire proteomes followed by multidimensional liquid-phase chromatographic (MDLC) separation of peptides, have been developed.13,14 In contrast to gel-based approaches, in shotgun/MDLC proteomics, the separation effort prior to MS analysis is focused on peptides. This approach may overcome limitations that are associated with the behavior of low abundant proteins and “difficult proteins” (e.g., membrane and r 2011 American Chemical Society
high molecular proteins) in protein separation methods. In the most common MDLC approach, proteolytic (tryptic) peptides are fractionated by strong cation exchange chromatography (SCX) in the first dimension.13,15 Due to the compatibility of the mobile phase with the electrospray ionization process and the outstanding peak capacity of reversed-phase columns for peptides, the preferred mode for the second dimension separation that is usually online coupled with the mass spectrometer is clearly RP chromatography. The two-dimensional separation, which is based on different retention mechanisms of SCX and RP chromatography, leads to an orthogonal separation system in which the total peak capacity P is the product of the peak capacity of each dimension (P = PSCX PRP).14 Even though the actual orthogonality and peak capacity of 2-D systems is often lower than expected, it could be shown that, in particular, off-line 2-D (SCX-RP) LC approaches are robust and flexible with total peak capacities of >10,000 that allow the analysis of highly complex mixtures of proteins, e.g., the tryptic digest of E. coli containing more than 4000 proteins.16 However, there are a few drawbacks: initially, the resolution and separation efficiency of SCX for peptides is limited, and second, the elution buffer contains a significant amount of salt that has to be removed before nanoLC-MS/MS.17 Received: September 7, 2011 Published: November 11, 2011 1175
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Figure 1. Principle of ADAP peptide pull-down. Proteins were labeled in cell culture with “light” or “heavy” Lys and Arg (SILAC). Phosphopeptides covering the phosphorylation sites Y595 and the corresponding nonphosphorylated analogues were covalently coupled to agarose beads. Phosphorylated and nonphosphorylated baits were incubated with the “heavy” and “light” labeled lysate, respectively.
A variety of 2-D separation systems have been developed that employ reversed-phase chromatography in both dimensions.18,19 RPRP systems using mobile phases with high pH in the first separation and low pH in the second dimension, coupled either online or offline to ESI-MS or MALDI-MS, respectively, show practical peak capacities that are comparable to the common SCX-RP approach.17,18,20,21 In 2-D RP-RP systems, the slightly reduced orthogonality in comparison to SCX-RP is compensated by the considerable higher resolution and peak capacity of the RP mode for peptides in the first dimension.21 Several proteomic studies have demonstrated that RP-RP approaches using two different pH values have a great potential for shotgun analysis of complex protein mixtures.20 24 Recently, a proteome analysis of S. cerevisiae whole digest was described that employs an off-line RP-RP nanoLC-MS/MS system with the same mobile phase and pH in both separation dimensions.25 The results show that this simple and non-orthogonal 2-D LC method enables a comprehensive analysis of proteomes of moderate complexity. In this work, we characterize peptide-protein interactions using an improved RP-RP LC-MS/MS approach. We perform a combination of peptide pull-down assay and SILAC-based relative protein quantification26 to identify potential interaction partners of site specifically phosphorylated protein sequences. To examine whether the 2-D RP-RP nanoLC-MS/MS system can be used for this kind of interactome analysis, we compare 2-D LC-MS/ MS and previously published GeLC-MS/MS data11 for the reliable identification of binding proteins and the unambiguous annotation of modification-specific peptide interactors.
’ EXPERIMENTAL SECTION Preparation of Peptide Baits
Tyr-595-phosphorylated ADAP sequence R586PIEDDQEVY(p)DDVAE600 and the corresponding unmodified peptide were synthesized by standard solid-phase peptide synthesis (Fmoc chemistry) and purified by preparative HPLC as described earlier.11,27 An N-terminal cysteine was added for covalent
binding to agarose beads. After lyophilization all peptides were shown to have >95% purity, according to HPLC profiles with photometric detection at 220 nm, and gave the expected mass peaks by ESI-TOF-MS. In order to form the affinity matrix, peptides were immobilized on agarose beads (SulfoLink, Pierce) as previously described.11 The peptide loading of all matrices was determined by quantitative amino acid analysis. SILAC Cell Culture and Peptide Pull-Down
Jurkat T cells (clone E6-1) were cultured in RPMI1640 SILAC medium containing 10% dialyzed FBS (SILAC quantification kit, Pierce) and either light arginine and lysine or heavy-labled 13 C6,15N4-arginine and 13C6-lysine (Silantes). Cells were cultured in “light” or “heavy” media for 8 days in a humified atmosphere with 5% CO2. Labeling efficiencies were determined by mass spectrometry of tryptic digests of selected 1DE-bands and were found to be >95%. “Light” and “heavy” labeled Jurkat T cells (∼2 107 each) were lysed in 2 100 μL of lysis buffer (10 mM Hepes (pH 7.5), 150 mM NaCl, 10 mM MgCl2, 10 mM KCl, 0.5 mM EGTA) with 1% (v/v) NP-40, 1 mM Na3VO4, protease inhibitor cocktail (complete, EDTA-free, Roche Applied Science, Mannheim, Germany) on ice for 30 min. The cell extracts were vortexed and centrifuged for 10 min at 8000g at 4 °C. Peptide binding assays were performed using a reverse labeling strategy (crossover experiments). Matrix-bound ADAP-peptides (modified and nonmodified) were incubated with either labeled or unlabeled T cell lysate. An equal amount of total protein (∼1.5 mg) was incubated with 20 μL of agarose beads (∼15 nmol of peptide) at 20 °C for 1 h. Beads were washed four times with lysis buffer to reduce nonspecific binding. On-Bead Digestion with Trypsin
Beads containing bound proteins from either light or heavy isotope containing lysates were combined directly after peptide pull-down and washing. Tryptic on-bead digestion was performed 1176
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Figure 2. 2-D LC-MS workflow. In the first dimension, fractions from sequential elution sections (10 to 12 min) were pooled (e.g., 1 + 17, 2 + 18, 3 + 19, ...). After reducing the amount of organic co-solvent (acetonitrile) in a vacuum centrifuge, the fractions were submitted to nanoLC-MS analysis using a C18 stationary phase. First dimension microLC was equipped with a polymeric 150 mm 1 mm column, 3 μm, 100 Å. Mobile phase A, 0.1% TFA, 5% acetonitrile in water; mobile phase B, 0.085% TFA, 80% acetonitrile in water; flow rate, 40 μL/min; linear gradient, 1 50% B in 50 min. Second dimension nanoLC-MS/MS was equipped with a 150 mm 75 μm Atlantis dC18 column, 3 μm, 100 Å. Mobile phase A, 0.1% formic acid in water; mobile phase B, 0.1% formic acid in acetonitrile; flow rate, 40 μL/min; linear gradient, 0 40% B in 90 min.
with 4 μg trypsin (sequencing grade modified, Promega) in 200 μL 50 mM ammonium bicarbonate for 17 h at 37 °C. The enzymatic reaction was terminated by adding 50 μL of 0.5% (v/v) trifluoroacetic acid in acetonitrile. After centrifugation, the supernatant was retained and beads were washed with 200 μL acetonitrile. Supernatants were combined and concentrated to 2 μL in a vacuum centrifuge. Samples were reconstituted in 90 μL of 0.1% (v/v) TFA, 5% (v/v) acetonitrile in water and subjected to the first chromatographic step. First Dimension RP Chromatography
Separation in the first dimension by microLC was carried out using an Dionex/LC Packings Ultimate HPLC System on a polymeric column (PLRP-S, 3 μm, 100 Å, 150 mm 1.0 mm i.d., Varian) at an eluent flow rate of 40 μL/min. A linear gradient of 1-50% B in 50 min was applied. Mobile phase A was 0.1% TFA, 5% acetonitrile in water; mobile phase B was 0.085% TFA, 80% acetonitrile in water. Eluent were collected each 0.5 or 0.75 min and combined to 16 or 24 fractions as shown in Figure 2. The samples were dried under vacuum and reconstituted in 6 μL of 0.1% (v/v) TFA, 5% (v/v) acetonitrile in water. For normalized retention time plots, separations were carried out on a polymeric column (PLRP-S, 3 μm, 100 Å, 150 mm 1.0 mm i.d., Varian) and PepMap C18 (5 μm, 100 Å, 150 mm 1 mm i.d., Dionex), respectively. The gradient was 1% B/min, flow rate was 40 μL/min. Mobile phase A was 0.1% TFA or formic acid, 5% acetonitrile in water; mobile phase B was 0.1% TFA or formic acid, 80% acetonitrile in water. Separations at high
pH were run with the polymeric PLRP-S column. Mobile Mobile phase A was 20 mM ammonium format (pH10), 5% acetonitrile in water; mobile phase B was 20 mM ammonium format (pH10), 80% acetonitrile in water. NanoLC-MS
LC-MS/MS analyses were performed on a LTQ-Orbitrap XL mass spectrometer (Thermo Scientific) equipped with an Eksigent 2D nanoflow LC system (Axel Semrau GmbH). The LC system was coupled to the mass spectrometer via a nanoelectrospray source (Proxeon) with a 10 μm i.d. PicoTip ESI emitter (New Objective). Six microliters of the sample were injected and concentrated on a trap column (PepMap C18, 5 μm, 100 Å, 5 mm 300 μm i.d., Dionex) and equilibrated with 0.1% TFA, 2% acetonitrile in water. After switching the trap column in-line, LC separations were performed on a capillary column (Atlantis dC18, 3 μm, 100 Å, 150 mm 75 μm i.d., Waters) at an eluent flow rate of 250 nL/min using a linear gradient of 0-40% B in 90 min. Mobile phase A contained 0.1% formic acid (v/v) in water; mobile phase B contained 0.1% formic acid in acetonitrile. Mass spectra were acquired in a data-dependent mode with one MS survey scan (with a resolution of 60,000) in the Orbitrap and MS/MS scans of the five most intense precursor ions in the LTQ. The MS survey range was m/z 350-1500. The dynamic exclusion time (for precursor ions) was set to 120 s and automatic gain control was set to 3 106 and 20,000 for Orbitrap-MS and LTQMS/MS scans, respectively. 1177
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Data Processing and Quantification
Identification and quantification of proteins were carried out with version 1.0.12.31 of the MaxQuant software package as previously described.28 In brief, generated peak lists (msm files) were submitted to a MASCOT search engine (version 2.2, Matrix Science Ltd., London) and searched against an IPI human protein database (version 3.52). The mass tolerance of precursor and sequence ions was set to 7 ppm and 0.35 Da, respectively. Methionine oxidation and the acrylamide modification of cysteine were used as variable modifications. False discovery rates were 20
6
>20
8
GRB2-related adapter protein 2
>20
10
>20
11
>20
15
7 20
>20 >20
12 15
>20 >20
15 21
>20
11 10
NCK1
P16333
Cytoplasmic protein NCK1
>20 >20 >20
22
>20
17
>20
20
NCK2
O43639
Cytoplasmic protein NCK2
>20
16
>20
3
>20
13
>20
17
>20
8
>20
12
PIK3R1
P27986
Phosphatidylinositol 3-kinase regulatory subunit alpha
PLCG1
P19174
23
11.1
5
8.3
8
>20
25
12.0
7
10.2
9
1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase γ-1
>20
44
>20
34
>20
40
>20 12.7
51 29
>20 >20
41 14
>20 >20
37 25
>20
28
>20
21
>20
23
SLP76
Q13094
Lymphocyte cytosolic protein 2
FER
P16591
Proto-oncogene tyrosine-protein kinase FER
RASA1
P20936
Ras GTPase-activating protein 1
10.5
6.7
8
6.1
5
5.4
9
16.8
3
17.8
4
6.4
10
8.2
8
11.0 >20
CRKL
P46109
CRKL Crk-like protein
4.7 >20
9
>20
8
16
>20
10
>20
7
>20
4
>20
8
16.1
7
16.4
8 6 5
a
Previously published data of SILAC-based pull-down experiments based on SDS-PAGE fractionation of proteins followed by nanoLC-MS/MS for quantification of tryptic peptides as described.11 b Number of quantified peptides were taken from the MaxQuant algorithm (razor peptides). Ratios of two independent pull-down experiments are listed.
to detect exactly the same potential binding partners of Tyr 595phosphorylated ADAP as detected in previous GeLC-MS/MS experiments. Both the total number of unique peptides used for identification and quantification and the specific binders found demonstrate the efficiency of the 2-D RP-RP LC-MS/MS approach.
’ CONCLUSION In this study, an improved offline 2-D LC-MS/MS approach for the identification and quantification of binding proteins in pulldown experiments has been developed, utilizing RP separations with acidic mobile phases in both LC dimensions. We have applied the approach to analyze the interactome of phosphorylated 1181
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Journal of Proteome Research peptide sequences of the T cell adapter protein ADAP.11 As a result of the use of different stationary and mobile phases in both dimensions, a sufficient orthogonality could be achieved. The high resolution in both RP-LC dimensions in combination with an optimized fractionation scheme allows the analysis of complex protein mixtures resulting from affinity pull-down experiments. Using quantitative SILAC/MS, peptide pull-downs with Tyr 595-phosphorylated ADAP peptides resulted in the identification of phosphorylation-specific protein interactions in the presence of an excess of nonspecific protein background (about 1,000 proteins). All of the potential binding partners of ADAP that were identified by previous SILAC/GeLC-MS/MS experiments were also detected by the new 2-D RP-RP LCMS/MS approach. GeLC-based pull-down experiments usually require nanoLC-MS/MS analysis of 35-40 samples. In contrast, the presented fractionation scheme results in 16-24 fractions that have to be analyzed. The method reduces the time of analysis and does not require any buffer exchange or desalting steps before LC-MS. The simple and robust offline 2-D RP-RP LC-MS/MS method presented here offers an attractive, time-saving alternative for reliable proteomic analysis, in particular if the complexity of the protein mixture is reduced to less than 2000-2500 proteins by a preceding affinity step as is typical in interaction studies.
’ ASSOCIATED CONTENT
bS
Supporting Information Lists of identified and quantified proteins of 2-D RP-RP LCMS/MS experiments. This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*Tel. +49 30 94793221. Fax +49 30 94793222. E-mail: ekrause@ fmp-berlin.de. Present Addresses §
Biocomputing Group, Freie Universit€at Berlin, Arnimallee 6, 14195 Berlin, Germany.
’ REFERENCES (1) Mallick, P.; Kuster, B. Proteomics: A Pragmatic Perspective. Nat. Biotechnol. 2010, 28, 695–709. (2) Ahrens, C. H.; Brunner, E.; Qeli, E.; Basler, K.; Aebersold, R. Generating and Navigating Proteome Maps Using Mass Spectrometry. Nat. Rev. Mol. Cell Biol. 2010, 11, 789–801. (3) Lasonder, E.; Ishihama, Y.; Andersen, J. S.; Vermunt, A. M. W.; Pain, A.; Sauerwein, R. W.; Eling, W. M. C.; Hall, N.; Waters, A. P.; Stunnenberg, H. G.; Mann, M. Analysis of the Plasmodium falciparum Proteome by High-Accuracy Mass Spectrometry. Nature 2002, 419, 537–542. (4) Schirle, M.; Heurtier, M. A.; Kuster, B. Profiling Core Proteomes of Human Cell Lines by One-Dimensional PAGE and Liquid Chromatography-Tandem Mass Spectrometry. Mol. Cell. Proteomics 2003, 2, 1297–1305. (5) Zwang, N. A.; Hoffert, J. D.; Pisitkun, T.; Moeller, H. B.; Fenton, R. A.; Knepper, M. A. Identification of Phosphorylation-Dependent Binding Partners of Aquaporin-2 Using Protein Mass Spectrometry. J. Proteome Res. 2009, 8, 1540–1554. (6) Ong, S. E.; Blagoev, B.; Kratchmarova, I.; Kristensen, D. B.; Steen, H.; Pandey, A.; Mann, M. Stable Isotope Labeling by Amino Acids
ARTICLE
in Cell Culture, SILAC, As a Simple and Accurate Approach to Expression Proteomics. Mol. Cell Proteomics 2002, 1, 376–386. (7) Graumann, J.; Hubner, N. C.; Kim, J. B.; Ko, K.; Moser, M.; Kumar, C.; Cox, J.; Scholer, H.; Mann, M. Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) and Proteome Quantitation of Mouse Embryonic Stem Cells to a Depth of 5,111 Proteins. Mol. Cell. Proteomics 2008, 7, 672–683. (8) Ross, P. L.; Huang, Y. N.; Marchese, J. N.; Williamson, B.; Parker, K.; Hattan, S.; Khainovski, N.; Pillai, S.; Dey, S.; Daniels, S.; Purkayastha, S.; Juhasz, P.; Martin, S.; Bartlet-Jones, M.; He, F.; Jacobson, A.; Pappin, D. J. Multiplexed Protein Quantitation in Saccharomyces Cerevisiae Using Amine-Reactive Isobaric Tagging Reagents. Mol. Cell. Proteomics 2004, 3, 1154–1169. (9) Engmann, O.; Campbell, J.; Ward, M.; Giese, K. P.; Thompson, A. J. Comparison of a Protein-Level and Peptide-Level Labeling Strategy for Quantitative Proteomics of Synaptosomes Using Isobaric Tags. J. Proteome Res. 2010, 9, 2725–2733. (10) Mirgorodskaya, O. A.; Kozmin, Y. P.; Titov, M. I.; Korner, R.; Sonksen, C. P.; Roepstorff, P. Quantitation of Peptides and Proteins by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Using O-18-Labeled Internal Standards. Rapid Commun. Mass Spectrom. 2000, 14, 1226–1232. (11) Lange, S.; Sylvester, M.; Schumann, M.; Freund, C.; Krause, E. Identification of Phosphorylation-Dependent Interaction Partners of the Adapter Protein ADAP Using Quantitative Mass Spectrometry: SILAC Vs O-18-Labeling. J. Proteome Res. 2010, 9, 4113–4122. (12) Washburn, M. P.; Wolters, D.; Yates, J. R. Large-Scale Analysis of the Yeast Proteome by Multidimensional Protein Identification Technology. Nat. Biotechnol. 2001, 19, 242–247. (13) Peng, J. M.; Elias, J. E.; Thoreen, C. C.; Licklider, L. J.; Gygi, S. P. Evaluation of Multidimensional Chromatography Coupled with Tandem Mass Spectrometry (LC/LC-MS/MS) for Large-Scale Protein Analysis: The Yeast Proteome. J. Proteome Res. 2003, 2, 43–50. (14) Motoyama, A.; Yates, J. R., III Multidimensional LC Separations in Shotgun Proteomics. Anal. Chem. 2008, 80, 7187–7193. (15) Nagele, E.; Vollmer, M.; Horth, P.; Vad, C. 2D-LC/MS Techniques for the Identification of Proteins in Highly Complex Mixtures. Expert Rev. Proteomics 2004, 1, 37–46. (16) Eeltink, S.; Dolman, S.; Swart, R.; Ursem, M.; Schoenmakers, P. J. Optimizing the Peak Capacity Per Unit Time in One-Dimensional and Off-Line Two-Dimensional Liquid Chromatography for the Separation of Complex Peptide Samples. J. Chromatogr. A 2009, 1216, 7368–7374. (17) Gilar, M.; Olivova, P.; Daly, A. E.; Gebler, J. C. Orthogonality of Separation in Two-Dimensional Liquid Chromatography. Anal. Chem. 2005, 77, 6426–6434. (18) Gilar, M.; Olivova, P.; Daly, A. E.; Gebler, J. C. Two-Dimensional Separation of Peptides Using RP-RP-HPLC System with Different pH in First and Second Separation Dimensions. J. Sep. Sci. 2005, 28, 1694–1703. (19) Toll, H.; Oberacher, H.; Swart, R.; Huber, C. G. Separation, Detection, and Identification of Peptides by Ion-Pair Reversed-Phase HighPerformance Liquid Chromatography-Electrospray Ionization Mass Spectrometry at High and Low pH. J. Chromatogr. A 2005, 1079, 274–286. (20) Nakamura, T.; Kuromitsu, J.; Oda, Y. Evaluation of Comprehensive Multidimensional Separations Using Reversed-Phase, ReversedPhase Liquid Chromatography/Mass Spectrometry for Shotgun Proteomics. J. Proteome Res. 2008, 7, 1007–1011. (21) Delmotte, N.; Lasaosa, M.; Tholey, A.; Heinzle, E.; Huber, C. G. Two-Dimensional Reversed-Phase x Ion-Pair Reversed-Phase HPLC: An Alternative Approach to High-Resolution Peptide Separation for Shotgun Proteome Analysis. J. Proteome Res. 2007, 6, 4363–4373. (22) Zhou, F.; Cardoza, J. D.; Ficarro, S. B.; Adelmant, G. O.; Lazaro, J. B.; Marto, J. A. Online Nanoflow RP-RP-MS Reveals Dynamics of Multicomponent Ku Complex in Response to DNA Damage. J. Proteome Res. 2010, 9, 6242–6255. (23) Lasaosa, M.; Delmotte, N.; Huber, C. G.; Melchior, K.; Heinzle, E.; Tholey, A. A 2D Reversed-Phase x Ion-Pair Reversed-Phase HPLC-MALDI 1182
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Journal of Proteome Research
ARTICLE
TOF/TOF-MS Approach for Shotgun Proteome Analysis. Anal. Bioanal. Chem. 2009, 393, 1245–1256. (24) Song, C. X.; Ye, M. L.; Han, G. H.; Jiang, X. N.; Wang, F. J.; Yu, Z. Y.; Chen, R.; Zou, H. F. Reversed-Phase-Reversed-Phase Liquid Chromatography Approach With High Orthogonality for Multidimensional Separation of Phosphopeptides. Anal. Chem. 2010, 82, 53–56. (25) Gokce, E.; Andrews, G. L.; Dean, R. A.; Muddiman, D. C. Increasing Proteome Coverage With Offline RP HPLC Coupled to Online RP NanoLC-MS. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 2011, 879, 610–614. (26) Ong, S. E.; Mann, M. A Practical Recipe for Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC). Nat. Protoc. 2006, 1, 2650–2660. (27) Klemm, C.; Schroder, S.; Gluckmann, M.; Beyermann, M.; Krause, E. Derivatization of Phosphorylated Peptides With S- and N-Nucleophiles for Enhanced Ionization Efficiency in Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry. Rapid Commun. Mass Spectrom. 2004, 18, 2697–2705. (28) Cox, J.; Mann, M. MaxQuant Enables High Peptide Identification Rates, Individualized P.p.b.-Range Mass Accuracies and ProteomeWide Protein Quantification. Nat. Biotechnol. 2008, 26, 1367–1372. (29) McCalley, D. V. Effect of Buffer on Peak Shape of Peptides in Reversed-Phase High Performance Liquid Chromatography. J. Chromatogr. A 2004, 1038, 77–84. (30) Spicer, V.; Yamchuk, A.; Cortens, J.; Sousa, S.; Ens, W.; Standing, K. G.; Wilkins, J. A.; Krokhin, O. V. Sequence-Specific Retention Calculator. A Family of Peptide Retention Time Prediction Algorithms in Reversed-Phase HPLC: Applicability to Various Chromatographic Conditions and Columns. Anal. Chem. 2007, 79, 8762–8768. (31) Apffel, A.; Fischer, S.; Goldberg, G.; Goodley, P. C.; Kuhlmann, F. E. Enhanced Sensitivity for Peptide-Mapping With Electrospray LiquidChromatography Mass-Spectrometry in the Presence of Signal Suppression Due to Trifluoroacetic Acid-Containing Mobile Phases. J. Chromatogr. A 1995, 712, 177–190. (32) Huber, C. G.; Premstaller, A. Evaluation of Volatile Eluents and Electrolytes for High-Performance Liquid Chromatography-Electrospray Ionization Mass Spectrometry and Capillary ElectrophoresisElectrospray Ionization Mass Spectrometry of Proteins - I. Liquid Chromatography. J. Chromatogr. A 1999, 849, 161–173. (33) Gropengiesser, J.; Varadarajan, B. T.; Stephanowitz, H.; Krause, E. The Relative Influence of Phosphorylation and Methylation on Responsiveness of Peptides to MALDI and ESI Mass Spectrometry. J. Mass Spectrom. 2009, 44, 821–831. (34) McCalley, D. V. Comparison of an Organic Polymeric Column and a Silica-Based Reversed-Phase for the Analysis of Basic Peptides by High-Performance Liquid Chromatography. J. Chromatogr. A 2005, 1073, 137–145. (35) Geng, L.; Raab, M.; Rudd, C. E. Cutting Edge: SLP-76 Cooperativity With FYB/FYN-T in the Up-Regulation of TCR-Driven IL-2 Transcription Requires SLP-76 Binding to FYB at Tyr595 and Tyr651. J. Immunol. 1999, 163, 5753–5757. (36) Raab, M.; Kang, H.; da, S. A.; Zhu, X.; Rudd, C. E. FYN-T-FYBSLP-76 Interactions Define a T-Cell Receptor Zeta/CD3-Mediated Tyrosine Phosphorylation Pathway That Up-Regulates Interleukin 2 Transcription in T-Cells. J. Biol. Chem. 1999, 274, 21170–21179. (37) Lettau, M.; Pieper, J.; Gerneth, A.; Lengl-Janssen, B.; Voss, M.; Linkermann, A.; Schmidt, H.; Gelhaus, C.; Leippe, M.; Kabelitz, D.; Janssen, O. The Adapter Protein Nck: Role of Individual SH3 and SH2 Binding Modules for Protein Interactions in T Lymphocytes. Protein Sci. 2010, 19, 658–669. (38) Sylvester, M.; Kliche, S.; Lange, S.; Geithner, S.; Klemm, C.; Schlosser, A.; Grossmann, A.; Stelzl, U.; Schraven, B.; Krause, E.; Freund, C. Adhesion and Degranulation Promoting Adapter Protein (ADAP) Is a Central Hub for Phosphotyrosine-Mediated Interactions in T Cells. PLoS One 2010, 5, e11708.
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