Online Nanoflow Multidimensional Fractionation for High Efficiency Phosphopeptide Analysis
2011; Elsevier BV; Volume: 10; Issue: 11 Linguagem: Inglês
10.1074/mcp.o111.011064
ISSN1535-9484
AutoresScott B. Ficarro, Yi Zhang, Marlene J. Carrasco-Alfonso, Brijesh K. Garg, Guillaume Adelmant, James T. Webber, Chance John Luckey, Jarrod A. Marto,
Tópico(s)Blood disorders and treatments
ResumoDespite intense, continued interest in global analyses of signaling cascades through mass spectrometry-based studies, the large-scale, systematic production of phosphoproteomics data has been hampered in-part by inefficient fractionation strategies subsequent to phosphopeptide enrichment. Here we explore two novel multidimensional fractionation strategies for analysis of phosphopeptides. In the first technique we utilize aliphatic ion pairing agents to improve retention of phosphopeptides at high pH in the first dimension of a two-dimensional RP-RP. The second approach is based on the addition of strong anion exchange as the second dimension in a three-dimensional reversed phase (RP)-strong anion exchange (SAX)-RP configuration. Both techniques provide for automated, online data acquisition, with the 3-D platform providing the highest performance both in terms of separation peak capacity and the number of unique phosphopeptide sequences identified per μg of cell lysate consumed. Our integrated RP-SAX-RP platform provides several analytical figures of merit, including: (1) orthogonal separation mechanisms in each dimension; (2) high separation peak capacity (3) efficient retention of singly- and multiply-phosphorylated peptides; (4) compatibility with automated, online LC-MS analysis. We demonstrate the reproducibility of RP-SAX-RP and apply it to the analysis of phosphopeptides derived from multiple biological contexts, including an in vitro model of acute myeloid leukemia in addition to primary polyclonal CD8+ T-cells activated in vivo through bacterial infection and then purified from a single mouse. Despite intense, continued interest in global analyses of signaling cascades through mass spectrometry-based studies, the large-scale, systematic production of phosphoproteomics data has been hampered in-part by inefficient fractionation strategies subsequent to phosphopeptide enrichment. Here we explore two novel multidimensional fractionation strategies for analysis of phosphopeptides. In the first technique we utilize aliphatic ion pairing agents to improve retention of phosphopeptides at high pH in the first dimension of a two-dimensional RP-RP. The second approach is based on the addition of strong anion exchange as the second dimension in a three-dimensional reversed phase (RP)-strong anion exchange (SAX)-RP configuration. Both techniques provide for automated, online data acquisition, with the 3-D platform providing the highest performance both in terms of separation peak capacity and the number of unique phosphopeptide sequences identified per μg of cell lysate consumed. Our integrated RP-SAX-RP platform provides several analytical figures of merit, including: (1) orthogonal separation mechanisms in each dimension; (2) high separation peak capacity (3) efficient retention of singly- and multiply-phosphorylated peptides; (4) compatibility with automated, online LC-MS analysis. We demonstrate the reproducibility of RP-SAX-RP and apply it to the analysis of phosphopeptides derived from multiple biological contexts, including an in vitro model of acute myeloid leukemia in addition to primary polyclonal CD8+ T-cells activated in vivo through bacterial infection and then purified from a single mouse. Reversible phosphorylation plays a central role in the regulation of normal cell physiology. The strong links between aberrant signaling and human disease, along with the potential for specific inhibition of disrupted kinase activity, continue to drive efforts aimed at systematic and large-scale analysis of phosphorylation in cells and tissues. Shortly after introduction of immobilized metal affinity chromatography (IMAC) 1The abbreviations used are: IMACimmobilized metal affinity chromatographyAMLAcute Myeloid LeukemiaCADcollisionally activated dissociationESIelectrospray ionizationFDRFalse Discovery RateFLFLT3 ligandFLT3FMS-like tyrosine kinase 3LC/MSliquid chromatography/mass spectrometryLm-OVARecombinant Listeria monocytogenes expressing chicken ovalbuminITDinternal tandem duplicationMS/MSmass spectrometry/mass spectrometry or tandem mass spectrometryNTAnitrilotetraacetic acidRP-RPreversed phase-reversed phaseRP-SAX-RPreversed phase-strong anion exchange-reversed phaseRTretention timeSCXstrong cation exchangeTFAtrifluoroacetic acidUPLCultra-high pressure liquid chromatographyWTwild type. as an enrichment tool prior to mass spectrometry (MS) analysis (1Nuwaysir L. Stults J. Electrospray ionization mass spectrometry of phosphopeptides isolated by on-line immobilized metal-ion affinity chromatography.J. Am. Soc. 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Over the past ca. 5 years, the performance of phosphopeptide enrichment protocols and related methods has stabilized; in fact several groups (15Dephoure N. Zhou C. Villén J. Beausoleil S.A. Bakalarski C.E. Elledge S.J. Gygi S.P. A quantitative atlas of mitotic phosphorylation.Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 10762-10767Crossref PubMed Scopus (1145) Google Scholar, 16Song C. Ye M. Han G. Jiang X. Wang F. Yu Z. Chen R. Zou H. Reversed-Phase-Reversed-Phase Liquid Chromatography Approach with High Orthogonality for Multidimensional Separation of Phosphopeptides.Anal. Chem. 2010; 82: 53-56Crossref PubMed Scopus (120) Google Scholar, 17Han G. Ye M. Zhou H. Jiang X. Feng S. Jiang X. Tian R. Wan D. Zou H. Gu J. Large-scale phosphoproteome analysis of human liver tissue by enrichment and fractionation of phosphopeptides with strong anion exchange chromatography.Proteomics. 2008; 8: 1346-1361Crossref PubMed Scopus (171) Google Scholar, 18Pinkse M.W. Mohammed S. 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Although these strategies provide for larger phosphosite catalogs, closer inspection reveals that the analytical efficiency, as measured by the number of phosphopeptide identifications per microgram of biological lysate consumed, has remained surprisingly consistent at ≈1–10 phosphopeptides/μg across a wide range of sample types (Table I). One explanation is that the physicochemical properties of phosphopeptides render them less amenable to fractionation by commonly used techniques. For example, although the combination of strong cation exchange (SCX) with reversed phase (RP) has been tremendously successful for fractionation of tryptic peptides generally (24Link A.J. Eng J. Schieltz D.M. Carmack E. Mize G.J. Morris D.R. Garvik B.M. Yates 3rd., J.R. Direct analysis of protein complexes using mass spectrometry.Nat. Biotechnol. 1999; 17: 676Crossref PubMed Scopus (1988) Google Scholar, 25Washburn M.P. Wolters D. Yates 3rd, J.R. 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Proteomics. 2004; 3: 1093-1101Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar) and low separation peak capacity (28Gilar 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. Separation Sci. 2005; 28: 1694-1703Crossref PubMed Scopus (352) Google Scholar). These results are consistent with the modest difference in pKa between the binding sites of typical SCX resins and phosphorylated side chains of serine, threonine, and tyrosine amino acids. These limitations have led to the development of numerous other strategies for fractionation of phosphopeptides including strong anion exchange chromatography (17Han G. Ye M. Zhou H. Jiang X. Feng S. Jiang X. Tian R. Wan D. Zou H. Gu J. Large-scale phosphoproteome analysis of human liver tissue by enrichment and fractionation of phosphopeptides with strong anion exchange chromatography.Proteomics. 2008; 8: 1346-1361Crossref PubMed Scopus (171) Google Scholar, 22Nühse T.S. Stensballe A. Jensen O.N. Peck S.C. Large-scale analysis of in vivo phosphorylated membrane proteins by immobilized metal ion affinity chromatography and mass spectrometry.Mol. Cell. Proteomics. 2003; 2: 1234-1243Abstract Full Text Full Text PDF PubMed Scopus (508) Google Scholar), continuous pH gradients (29Dai J. Wang L.S. Wu Y.B. Sheng Q.H. Wu J.R. Shieh C.H. Zeng R. Fully automatic separation and identification of phosphopeptides by continuous pH-gradient anion exchange online coupled with reversed-phase liquid chromatography mass spectrometry.J. Proteome Res. 2008; 8: 133-141Crossref Scopus (56) Google Scholar), ERLIC (30Alpert A.J. Electrostatic repulsion hydrophilic interaction chromatography for isocratic separation of charged solutes and selective isolation of phosphopeptides.Anal. Chem. 2008; 80: 62-76Crossref PubMed Scopus (410) Google Scholar, 31Gan C.S. Guo T. Zhang H. Lim S.K. Sze S.K. A Comparative Study of Electrostatic Repulsion-Hydrophilic Interaction Chromatography (ERLIC) versus SCX-IMAC-Based Methods for Phosphopeptide Isolation/Enrichment.J. Proteome Res. 2008; 7: 4869-4877Crossref PubMed Scopus (87) Google Scholar), and hydrophilic interaction chromatography (19McNulty D.E. Annan R.S. Hydrophilic interaction chromatography reduces the complexity of the phosphoproteome and improves global phosphopeptide isolation and detection.Mol. Cell. Proteomics. 2008; 7: 971-980Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar), all utilizing RP chromatography for the second dimension separation. In this report we describe two approaches for fractionation of phosphopeptides that build upon our recent work (32Zhou 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: 6242-6255Crossref PubMed Scopus (32) Google Scholar) in coupling true, nanoflow chromatography with reversed phase-reversed phase (RP-RP) fractionation (28Gilar 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. Separation Sci. 2005; 28: 1694-1703Crossref PubMed Scopus (352) Google Scholar). First we utilize aliphatic-functionalized quarternary amines as ion pairing agents to improve retention of phosphopeptides in the first dimension RP separation performed at high pH. In a second approach, we use the standard ammonium formate RP-RP buffer system (28Gilar 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. Separation Sci. 2005; 28: 1694-1703Crossref PubMed Scopus (352) Google Scholar), and add anion exchange as a second dimension in a 3-D, RP-SAX-RP configuration. The latter provided for the highest performance in terms of separation peak capacity, orthogonality, and the number of unique phosphopeptide sequences identified per μg of cell lysate consumed. We demonstrate the technical reproducibility of RP-SAX-RP at various fractionation depths and input levels. Finally, we use RP-SAX-RP for the quantitative analysis of divergent signaling between two clinically relevant, constitutively active FLT3 mutants in an in vitro model of acute myeloid leukemia, in addition to qualitative identification of phosphopeptides in primary polyclonal CD8+ T-cells activated in vivo through bacterial infection and then purified from a single mouse. Collectively our RP-SAX-RP platform provides significantly improved efficiency (IDs/microgram input) along with multiple analytical figures of merit, including: (1) orthogonal separation mechanisms in each dimension; (2) high separation peak capacity; (3) efficient retention of singly- and multiply phosphorylated pepitdes; (4) compatibility with automated, online LC-MS analysis.Table IA survey of recent large scale phosphoproteomics studies. This list is non-comprehensive and some subsets of data were not readily availableReferenceSourceStarting materialPhospho peptidesIDs/μgPhosphorylation sitesSites/μg(112Pan C. Olsen J.V. Daub H. Mann M. Global effects of kinase inhibitors on signaling networks revealed by quantitative phosphoproteomics.Mol. Cell. Proteomics. 2009; 8: 2796-2808Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar)K562, Hela20 mg56890.350640.3(15Dephoure N. Zhou C. Villén J. Beausoleil S.A. Bakalarski C.E. Elledge S.J. Gygi S.P. A quantitative atlas of mitotic phosphorylation.Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 10762-10767Crossref PubMed Scopus (1145) Google Scholar)Hela12 mg683795.7142651.2(113Bodenmiller B. Mueller L.N. Mueller M. Domon B. Aebersold R. Reproducible isolation of distinct, overlapping segments of the phosphoproteome.Nature Methods. 2007; 4: 231-237Crossref PubMed Scopus (0) Google Scholar)Drosoph1.5 mg100976.7bDenotes detected features after enrichment.(8Ficarro S.B. Adelmant G. Tomar M.N. Zhang Y. Cheng V.J. Marto J.A. Magnetic bead processor for rapid evaluation and optimization of parameters for phosphopeptide enrichment.Anal. Chem. 2009; 81: 4566-4575Crossref PubMed Scopus (113) Google Scholar)K562100 μg110011.0(114Van Hoof D. Muñoz J. Braam S.R. Pinkse M.W. Linding R. Heck A.J. Mummery C.L. Krijgsveld J. Phosphorylation dynamics during early differentiation of human embryonic stem cells.Cell Stem Cell. 2009; 5: 214-226Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar)hESC1 mg30903.130673.1(115Swaney D.L. Wenger C.D. Thomson J.A. Coon J.J. Human embryonic stem cell phosphoproteome revealed by electron transfer dissociation tandem mass spectrometry.Proc. Natl. Acad. Sci. U. S. A. 2009; 106: 995-1000Crossref PubMed Scopus (164) Google Scholar)hESC10 mg519205.2626426.3(96Olsen J.V. Vermeulen M. Santamaria A. Kumar C. Miller M.L. Jensen L.J. Gnad F. Cox J. Jensen T.S. Nigg E.A. Brunak S. Mann M. Quantitative phosphoproteomics reveals widespread full phosphorylation site occupancy during mitosis.Sci. Signal. 2010; 3: ra3Crossref PubMed Scopus (1000) Google Scholar)HeLa10 mg247142.5(69Choudhary C. Olsen J.V. Brandts C. Cox J. Reddy P.N. Böhmer F.D. Gerke V. Schmidt-Arras D.E. Berdel W.E. Müller-Tidow C. Mann M. Serve H. Mislocalized activation of oncogenic RTKs switches downstream signaling outcomes.Mol. Cell. 2009; 36: 326-339Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar)32D10 mg147001.5(116Mayya V. Lundgren D.H. Hwang S.I. Rezaul K. Wu L. Eng J.K. Rodionov V. Han D.K. Quantitative phosphoproteomic analysis of T cell receptor signaling reveals system-wide modulation of protein-protein interactions.Sci. Signal. 2009; 2: ra46Crossref PubMed Scopus (281) Google Scholar)Jurkat10 mg117081.2106651.1(117Brill L.M. Xiong W. Lee K.B. Ficarro S.B. Crain A. Xu Y. Terskikh A. Snyder E.Y. Ding S. Phosphoproteomic analysis of human embryonic stem cells.Cell Stem Cell. 2009; 5: 204-213Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar)hESC2 mg25481.3(118Malik R. Lenobel R. Santamaria A. Ries A. Nigg E.A. Körner R. Quantitative analysis of the human spindle phosphoproteome at distinct mitotic stages.J. Proteome Res. 2009; 8: 4553-4563Crossref PubMed Scopus (93) Google Scholar)HeLa400 μg19404.9(119Pan C. Gnad F. Olsen J.V. Mann M. Quantitative phosphoproteome analysis of a mouse liver cell line reveals specificity of phosphatase inhibitors.Proteomics. 2008; 8: 4534-4546Crossref PubMed Scopus (87) Google Scholar)Hepa1–610 mg54330.5(120Holt L.J. Tuch B.B. Villén J. Johnson A.D. Gygi S.P. Morgan D.O. Global analysis of Cdk1 substrate phosphorylation sites provides insights into evolution.Science. 2009; 325: 1682-1686Crossref PubMed Scopus (587) Google Scholar)Yeast10 mg740937.4106561.1(121Han G. Ye M. Zhou H. Jiang X. Feng S. Jiang X. Tian R. Wan D. Zou H. Gu J. Large-scale phosphoproteome analysis of human liver tissue by enrichment and fractionation of phosphopeptides with strong anion exchange chromatography.Proteomics. 2008; 8: 1346-1361Crossref PubMed Scopus (171) Google Scholar)LiveraDenotes analysis of primary tissue.1 mg3050.32740.3(122Xia Q. Cheng D. Duong D.M. Gearing M. Lah J.J. Levey A.I. Peng J. Phosphoproteomic analysis of human brain by calcium phosphate precipitation and mass spectrometry.J. Proteome Res. 2008; 7: 2845-2851Crossref PubMed Scopus (68) Google Scholar)BrainaDenotes analysis of primary tissue.5 mg5510.14660.1(123Zahedi R.P. Lewandrowski U. Wiesner J. Wortelkamp S. Moebius J. Schütz C. Walter U. Gambaryan S. Sickmann A. Phosphoproteome of resting human platelets.J. Proteome Res. 2008; 7: 526-534Crossref PubMed Scopus (132) Google Scholar)PlateletsaDenotes analysis of primary tissue.5 mg6300.16740.1(110Carrascal M. Ovelleiro D. Casas V. Gay M. Abian J. Phosphorylation analysis of primary human t lymphocytes using sequential imac and titanium oxide enrichment.J. Proteome Res. 2008; 7: 5167-5176Crossref PubMed Scopus (62) Google Scholar)TcellsaDenotes analysis of primary tissue.4 mg2530.12810.1(124Højlund K. Bowen B.P. Hwang H. Flynn C.R. Madireddy L. Geetha T. Langlais P. Meyer C. Mandarino L.J. Yi Z.P. In vivo Phosphoproteome of human skeletal muscle revealed by phosphopeptide enrichment and HPLC-ESI-MS/MS.J. Proteome Res. 2009; 8: 4954-4965Crossref PubMed Scopus (64) Google Scholar)MuscleaDenotes analysis of primary tissue.3 mg8790.3(125Wiśniewski J.R. Nagaraj N. Zougman A. Gnad F. Mann M. Brain phosphoproteome obtained by a FASP-based method reveals plasma membrane protein topology.J. Proteome Res. 2010; 9: 3280-3289Crossref PubMed Scopus (205) Google Scholar)BrainaDenotes analysis of primary tissue.3 mg80142.7(126Thingholm T.E. Jensen O.N. Robinson P.J. Larsen M.R. SIMAC (sequential elution from IMAC), a phosphoproteomics strategy for the rapid separation of monophosphorylated from multiply phosphorylated peptides.Mol. Cell. Proteomics. 2008; 7: 661-671Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar)hMSC120 μg4924.17166.0(127Thingholm T.E. Larsen M.R. Ingrell C.R. Kassem M. Jensen O.N. TiO2-based phosphoproteomic analysis of the plasma membrane and the effects of phosphatase inhibitor treatment.J. Proteome Res. 2008; 7: 3304-3313Crossref PubMed Scopus (0) Google Scholar)hMSC PM100 μg7577.67037.0(19McNulty D.E. Annan R.S. Hydrophilic interaction chromatography reduces the complexity of the phosphoproteome and improves global phosphopeptide isolation and detection.Mol. Cell. Proteomics. 2008; 7: 971-980Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar)Hela300 μg8142.710043.3a Denotes analysis of primary tissue.b Denotes detected features after enrichment. Open table in a new tab immobilized metal affinity chromatography Acute Myeloid Leukemia collisionally activated dissociation electrospray ionization False Discovery Rate FLT3 ligand FMS-like tyrosine kinase 3 liquid chromatography/mass spectrometry Recombinant Listeria monocytogenes expressing chicken ovalbumin internal tandem duplication mass spectrometry/mass spectrometry or tandem mass spectrometry nitrilotetraacetic acid reversed phase-reversed phase reversed phase-strong anion exchange-reversed phase retention time strong cation exchange trifluoroacetic acid ultra-high pressure liquid chromatography wild type. All multiplierz scripts referenced in the manuscript are freely available on our website at:http://blais.dfci.harvard.edu/index.php?id = 64. Magnetic Ni-NTA-agarose was obtained from Qiagen (Valencia, CA). Acetonitrile, EDTA, FeCl3, urea, and ammonium bicarbonate were from Sigma-Aldrich (St. Louis, MO). Trifluoroacetic acid was obtained from Pierce (Rockford, IL). Phosphopeptides EEpSGpSpSEEEAVLQR and LIEDAEpYTAK were synthesized using Fluorenylmethyloxycarbonyl chemistry. K562 cells were cultured in RPMI 1640 media supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37 °C in 5% CO2. Cells (≈5e7) were harvested by centrifugation during log phase. After washing twice with 20 ml phosphate buffered saline, the pellet was lysed with 3 ml of 8 m urea, 100 mm ammonium bicarbonate, and 30 μl each of Sigma-Aldrich phosphatase inhibitor cocktails I and II. Protein concentration was determined using the Bradford Assay (Bio-Rad laboratories, Hercules, CA). Proteins were reduced by adding dithiothreitol to a final concentration of 10 mm and incubating for 30 min at 60 °C, and alkylated with iodoacetamide (final concentration 20 mm) for 30 min in the dark at room temperature. Excess iodoacetamide was quenched by the addition of dithiotreitol to a final concentration of 20 mm. This solution was diluted to a final volume of 12 ml in 0.1 m ammonium bicarbonate. Trypsin (150 μg, 1:50 enzyme:substrate) was added and digestion was performed at 37 °C overnight. The resulting peptide solution was acidified with 10% trifluoroacetic acid (TFA), desalted on a C18 solid phase extraction cartridge and eluted with 25% acetonitrile with 0.1% TFA. Aliquots of eluted peptides were lyophilized by vacuum centrifugation and stored at −80 °C. Magnetic Ni-NTA agarose beads were washed 3× with 400 μl water, and treated with 400 μl of 100 mm EDTA, pH 8.0 for 30 min with end-over-end rotation. EDTA solution was removed, and beads were then washed 3× with 400 μl water, and treated with 600 μl of 10 mm aqueous FeCl3 solution for 30 min with end-over-end rotation. After removing excess metal ions, beads were washed 3× with 400 μl water, and 1× with 400 μl 1:1:1 acetonitrile:methanol:0.01% acetic acid. Tryptic peptides (in 80% MeCN/0.1% TFA; typical peptide concentrations were ≤1 μg/μl; see Supplemental Experimental Procedures for additional details) were then added to the beads, and phosphopeptide capture proceeded for 30 min with end-over-end rotation. After removing the supernatant, beads were washed 3× with 400 μl 80% acetonitrile/0.1% TFA. Phosphopeptides were eluted with 50 μl 1:20 ammonia/water for 30 min, dried to ≈5 μl by vacuum centrifugation, and reconstituted with 20 mm ammonium formate buffer. The multidimension fractionation platform (Fig. 1) consisted of a two-pump UPLC system with autosampler (Waters, Milford, MA) and an external valve (Valco, Austin, TX). For RP-RP experiments (Fig. 1A), the first dimension analytical column consisted of a 150 μm I.D. capillary packed with 5 cm of 5 μm C18 (XBridge, Waters). The weak needle wash and isocratic pump delivered various ion pairing agents all at pH 10.0. To transfer peptides between the first and second dimension columns, the trapping and vent valves were positioned to direct flow to the second dimension precolumn (150 μm I.D. capillary packed with 4 cm of POROS10R2, Applied Biosystems, Framingham, MA), the isocratic pump delivered 1 μl/min of pH 10 buffer (with sample loop of 20 μl in-line), and the binary pump delivered 10 μl/min of 0.2 m acetic acid (to dilute organic content and acidify the first dimension column effluent) for 25.5 min. Elution of peptides was accomplished by injection of acetonitrile fractions at pH 10.0 with or without an ion-pairing agent. After trapping, both valve positions were switched, to create a precolumn split and allow for gradient elution (2–30% B in 60 min, A = 0.2 m acetic acid, B = acetonitrile with 0.2 m acetic acid) of peptides to the analytical column (30 μm I.D. capillary packed with 12 cm of 5 μm Monitor C18, Column Engineering, Ontario, CA containing integrated 1 μm emitter tip), and into the mass spectrometer (Fig. 1B) at a flow rate of ≈30 nL/min. Three-dimensional RP-SAX-RP experiments were conducted in a similar fashion, except that an additional anion exchange column (150 μm I.D. capillary packed with 5 cm of POROS10HQ) was connected to the o
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