A Quantitative Analysis of Arabidopsis Plasma Membrane Using Trypsin-catalyzed 18O Labeling

• DOI: 10.1074/mcp.m500414-mcp200
• Publication Date: 2006-04-25T00:16:50Z
• Authors: Clark J. Nelson; Adrian D. Hegeman; Amy C. Harms; Michael R. Sussman
• Keywords: Quantitative Analysis; Isotopic labeling; Isobaric labeling

Typical mass spectrometry-based protein lists from purified fractions are confounded by the absence of tools for evaluating contaminants. In this report, we compare results a standard survey experiment using an ion trap spectrometer with those obtained dual isotope labeling and Q-TOF to quantify degree enrichment proteins in subcellular Arabidopsis plasma membrane. Incorporation stable isotope, either H218O or H216O, during trypsinization allowed relative quantification within membranes after phase partitioning polyethylene glycol/dextran mixtures. The ratios 174 membrane-associated 70 showing membrane equal greater than ATP-dependent proton pumps, canonical proteins. Enriched included several hallmark proteins, such as H+-ATPases, aquaporins, receptor-like kinases, various transporters, well number unknown functions. Most importantly, comparison datasets sequencing "survey" analysis that quantitative ratio method indicates many one-fourth putative identifications biological contaminants rather bona fide use tandem MS identification organelles compartments has expanded rapidly recent years (1Prime T.A. Sherrier D.J. Mahon P. Packman L.C. Dupree A proteomic thaliana.Electrophoresis. 2000; 16: 3488-3499Crossref Scopus (106) Google Scholar, 2Santoni V. Kieffer S. Desclaux D. Masson F. Rabilloud T. Membrane proteomics: additive main effects multiplicative interaction model classify according their solubility electrophoretic properties.Electrophoresis. 21: 3329-3344Crossref PubMed (211) 3Santoni Molloy M. un amour impossible?.Electrophoresis. 1054-1070Crossref (827) 4Santoni Rouquie Doumas Mansion Boutry Degand H. L. J. Prime Bauw G. Posada E. Rouze Dehais Sahnoun I. 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This technology reduces problems associated wide dynamic range abundances while also making it easier draw conclusions regarding significance. identified via sequence often generated studies, but is difficult assign observations legitimate elements specific Even if observation fide, unclear whether exclusively located given compartment cell. Application some form quantitation across purification schemes clearly necessary increase confidence organellar assignments.Most prior studies have relied on gel electrophoresis separation Although capable separating hundred once, significant limitations, particularly basic (3Santoni 15Gygi S.P. Corthals G.L. Zhang Y. Rochon Aebersold Evaluation electrophoresis-based technology.Proc. Natl. Acad. Sci. U. 97: 9390-9395Crossref (1200) Difficulties resolving identifying isoforms integral (PIPs) 1The abbreviations used are: PIP, protein; AHA, H+-ATPase; FP, false positive; LOPIT, localization tagging; RLK, kinase; SCX, strong cation exchange; vATPase, vacuolar type 2D, two-dimensional; ER, endoplasmic reticulum; TAIR, Information Resource; PPase, pyrophosphatase; GPI, glycosylphosphatidylinositol; DREPP, developmentally regulated polypeptide; CPK, calcium-dependent PLC, phospholipase C; PLD, D.1The H+-ATPases (AHAs), which both very multitransmembrane domain-containing polypeptide families, serve primary example limitation (2Santoni An alternative approach avoided isoelectric focusing polytopic one-dimensional SDS-PAGE separations (6Alexandersson successfully hundreds including these only validated purity preparations enzyme assays Western blots handful marker obvious weakness contamination issue discussed previously. As gel-based methods, shotgun uses LC fractionation. method, peptides tryptic digests fractionated exchange (SCX) then subjected reverse-phase MS. proven quite effective, producing thousands single (16Peng Elias Thoreen C.C. Licklider L.J. Gygi multidimensional chromatography coupled spectrometry (LC/LC-MS/MS) large-scale analysis: yeast proteome.J. 43-50Crossref (1371) 17Wolters Washburn M.P. Yates III, J.R. Anal. Chem. 2001; 73: 5683-5690Crossref (1557) One compared off-line 2D fractionation scheme chloroplast comparison, 283 were approach, whereas 243 further validating technique (13Froehlich Scholar).Stable isotopic conjunction attractive and, will demonstrate, provides facile means familiar version commercially available ICAT reagent (18Gygi Rist B. Gerber S.A. Turecek Gelb Nat. Biotechnol. 17: 994-999Crossref (4321) one investigation, Dunkley et al. (14Dunkley Scholar) applied density centrifugation total membranes. They labeled adjacent gradient, developing series By applying multivariate techniques comparing they novel Golgi reticulum (ER) components. referred (LOPIT). similar was validation rat liver (19Jiang X.S. Dai Sheng Q.H. Xia Q.C. Wu Zeng comparative research: bioinformatics prediction ascertain indication catalase.Mol. 12-34Abstract (73) Scholar).Alhough successful limited containing cysteines. Many few any cysteines, fragments may not be proper size spectral analysis. serine proteases trypsin incorporate two 18O atoms into carboxyl termini cleaved (20Yao X. Freas Ramirez Demirev P.A. Fenselau Proteolytic serotypes adenovirus.Anal. 2836-2842Crossref (774) 21Heller Mattou Menzel Yao Trypsin catalyzed 16O-to-18O MALDI-TOF, ESI-QTOF, ESI-ion spectrometers.J. Am. Soc. 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Using trypsin-catalyzed ESI-MSMS quantitation, fraction quantified enrichment. shows one-sixth data different thus represent Consistent role transport transduction, gene ontology predictions indicated transporters kinases functional categories sequenced origin.RESULTSAlthough does sufficient resolution small shifts spectrometer, shorter duty cycle therefore larger typical To assess sample potential possible, first conducted three independent analyses spectrometer. enrichment, upper lower phases two-phase-partitioned samples (Fig. 1). LOPIT Due our choice labels, analyzed because advantage offered spectrometer.False Positives—Multiple documented variability ability MS/MS search engines accurately through positives negatives 33Qian W.J. Liu Monroe M.E. Strittmatter E.F. Jacobs J.M. Kangas Petritis Camp II, D.G. Smith R.D. Probability-based SEQUEST human 53-62Crossref (294) 39Cargile B.J. Bundy Stephenson Jr., Potential positive large databases spectrometry.J. 1082-1085Crossref (173) False rates found function variables complexity, handling, charge state, platform, size, program used. Mascot attempt provide probabilistic estimate identification, surprising perform less perfectly (39Cargile 40MacCoss M.J. Computational data.Curr. Opin. Biol. 9: 88-94Crossref (49) empirical effective reverse strategy, spectra interest, known forward database, database. contains every reversed. Because same amino acid content, expected "chance" hits Scholar).Because operated mode where preference set doubly charged peptides, singly observed. result, 1% FP cutoff scores calculated triply 2A). had cutoffs 29 39, respectively, Mascot. For data, thresholds presented Fig. 2B. threshold 37, 21 18, respectively.Fig. 2.False rates.A, surveys Plots score versus shown. B, singly, doubly, isotopically samples. Pos., positive.View Image Figure ViewerDownload Hi-res image Download (PPT)MS Surveys—Samples digested aqueous/methanol solutions, resulting separated SCX. Each processed separately criteria described above. After combining identifications, there made 1016 unique locus genome. list provided Supplemental Table 1. Of 92 identifications. peptide, annotated 139 observed analyses, 205 3A). best knowledge, date membrane.Fig. 3.Overlap 18O-labeled samples.A, surveys, identified. From identified, analyses. shown overlap between second experiments experiments.View (PPT)Many residents AHAs, PIPs, (RLKs). Several other interest non-RLKs, G-proteins subunits, However, among would co-purify Some unexpected cytochrome b5, porins, ribosomal pyrophosphatase (PPase), H+-ATPase (vATPase) subunits. These mitochondrial, soluble, been (4–7). Whether membrane-localized simply hyperabundant species readily apparent simple surveys.Isotopic Labeling—In experiment, membrane-containing 18O-enriched water, endomembrane natural abundance water. labels reversed experiment. here text, reported normalized so phase-enriched always values one.Using above 116 Peptides 2. Between experiments, quantified. 38 peptide. spectrum describe 4. remaining dataset datasets, numbers each dataset. 3B.Fig. 4.MS/MS spectrum. fragmentation pattern AAEDTPPATASSDSSSTTAAAAPAK parent 1140.56 (a peptide) photosystem I subunit (At2g20260). 104.View (PPT)In AHA classified membrane-enriched. considering independently, biases avoided. total, showed Those characterized multiple I, II. log2 transform distributions 5). reference measurements, vanadate-sensitive ATPase assay results, measure abundance, 6A experiments.Table IPlasma peptidesGeneAnnotationTDsSc 1P1Q1Ratio 1Sc 2P2Q2Ratio 2At1g01620PIP1.361783312.13 ± 9.641462232.84 33.85At1g03870FLA9GPI————1042211.70 7.23At1g05150EF hand070217.251292212.63 0.47At1g19870Calmod.-bind088114.171072117.69At1g22530SEC1404441065.35 0.93671131111.22 2.10At1g30360ERD49396924.85 1.3139410310.48 2.74At1g48480LRR Kinase1————732110.34At1g59870PDR8144811054.90 1.963