Protein characterization by mass spectrometry C7250 Part IV Zbyněk Zdráhal RG Proteomics, CEITEC-MU Proteomics CF, CEITEC-MU NCBR FS MU zdrahal@sci.muni.cz Functional Genomics and Proteomics National Centre for Biomolecular Research Faculty of Science Masaryk University mu1 GIGO Appropriate sample preparation – key stone of success conservation of original protein status conservation of modification status (e.g. phosphatase inhibitors) removal of contaminants interferring MS analysis ... C7250 Demandingness of proteome analysis * protein number exceeds substantially number of genes human genome contains ~21 000 genů, but human proteome might contain ~ 1 000 000 proteins and their forms (isoforms, PTMs) (http://www.expasy.org/sprot/hpi/hpi_desc.html) ~ 10 000 proteins in cell * wide range of protein concentrations (~ 10 orders) necessity of efficient separation of high abundant components from low abundant ones, no PCR-like amplification method * wide range of physico-chemical properties * protein complexes necessity of protein complex analysis for deeper understanding mechanisms of cellular processes about 80% of proteins perform their funcions only as a part of a complex MM900282753[1] C7250 proteoforms C7250 ~ 105 peptides Direct LC-MS/MS analysis of the whole sample 1000 peptides/peak ~ 100 peptides ~ 900 peptides not measured scan rate time limitation Fractionation/separation to obtain maximum information C7250 Fractionation/separation the aim: to simplify extremely complex mixture to separate specific group of proteins/peptides (e.g. phosphopeptides) necessity of combination of separation principles – multidimensional separation selection of appropriate combination for given experiment (separation dimension might be also selected method of MS analysis) electrophoretic techniques: * isoelectric focusing (in-gel, in-liquid) * SDS PAGE * 2D gel electrophoresis (DIGE) * capillary electrophoresis chromatographic techniques: * liquid chromatography - reverse phase - ionex - molecular sieve - affinity (IMAC, MOAC, antibody) - HILIC (hydrophilic interaction chromatography) * off-line * on-line immunoprecipitation C:\Jaś\Oracle\Heart French New Caledonia.jpg Standard 1-D approaches 1-D GE protein digestion separation protein level MS/MS separation peptide level protein digestion MALDI-MS/MS LC-MS/MS „SIMPLE“ MIXTURES 1-D LC-MS/MS shotgun proteomics C7250 separation protein level MS MS/MS top-down high resolution !! Protein isolate of bacteriophage – 1-D separation Iden. – 5 major proteins LC-MS/MS kDa 66.2 45.0 31.0 21.5 14.4 97.4 GE cca 50 bands LOGO concentration range reduced possibility of minor component detection it infuenced by instrumentation LC separation conditions C7250 C:\Jaś\Oracle\pamutgy r.jpg 2-D GE 2-D LC-MS/MS protein digestion separation protein level MS/MS separation peptide level protein digestion Classical 2-D approaches 2-D LC-MS/MS MALDI-MS MALDI-MS/MS on-line/off-line LC-MALDI C7250 fag 812-Ag 2006-08-18 2-D GE/MALDI-MS Protein isolate of bacteriophage – 2-D separation ≈ 700 spots ≈ 20 proteins LOGO C7250 Protein mix digestion 2D - RP MS/MS 1D - SCX On-line (“MudPIT”) fractionation step by step 2D - RP MS/MS Peptide fraction 1D - SCX Off-line UV Peptide fraction Peptide fraction Peptide fraction Peptide fraction Peptide fraction Peptide fraction Peptide fraction LC 2-D LC peptides MudPIT (multidimensional protein identification technology) C7250 2-D LC peptides C7250 2-D LC (peptides) 1D-RP 2D-RP, fraction 5 mM 2D-RP, fraction 50 mM 2D-RP, fraction 100 mM Dionex application note C7250 2-D LC (peptides) RP-RP the same stationary phase – jiné pH www.ace-hplc.com Orthogonality of Separation C7250 Characterization of proteome and phosphoproteome of HEK293 cells lysis SDT buffer [SDS+DTT+Tris] HEK293 SPL – „scheduled precursor list“ analysis enables repeated analysis of sample with exclusion of already identified peptides in previous analysis 16 TiO2 enrichment analysis of phosphofractions LC-MS/MS (pH 3) (Orbitrap Elite) Data processing Data processing direct analysis w/o and with SPL LC-MS/MS (pH 3) (Orbitrap Elite) reversed phase pH 10 1D LC separation Data processing analysis of fractions LC-MS/MS (pH 3) (Orbitrap Elite) FASP TMT labeling cooperation with Assoc. Prof. Bryja group, FS MU C7250 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 LC-separation of the digested sample in 1D (high pH) 17 reversed phase pH 10 1D LC separation C7250 18 17931 20395 86212 95781 Number of identified peptides C7250 Data processing analysis of fractions LC-MS/MS (pH 3) (Orbitrap Elite) Number of identified proteins 9013 3398 3098 5474 5140 1317 1126 8560 Fractionation three fold increase in number of identified proteins Higher sequence coverage for most proteins C7250 Diameter of HPLC column vs sensitivity „Sensitivity increases with a decrease in column diameter because the same sample mass (amount) is eluted in a smaller volume. Therefore the concentration of the eluting peak is higher and the detection signal is stronger.“ Amer. Lab., 2001, 33 (10), 26-38. C7250 C7250 Capillary and nano columns * Increase in sensitivity * reduction of injected sample amounts * reduced consumption of solvents www.ace-hplc.com sorbents 1-D: ionex reverse phase HILIC IMAC (phospho) affinity (e.g. lectin – glyco) 2-D LC peptides On-line vs Off-line automation flexibility optimalization continuous collection of fractions C7250 2-D: reverse phase LC –MALDI (peptides) Peptide fraction nano LC (RP) ESI-MS/MS on-line MALDI TOF/TOF MS off-line Deposition on MALDI target Sample storage C7250 C:\Jaś\Oracle\Nouakchott Mauritania.jpg LC separation of complex protein mixtures Protein mix Protein 1D fraction Fractionation I. Ionex, SEC, …. Fractionation II. Protein 1D fraction Protein 2D fraction RP exp. exp. Protein 2D fraction digestion 1D (2D) HPLC ESI-MS/MS C7250 C7250 LC separation of complex protein mixtures Combination of GE a LC separation slices protein fractionation digestion nano 1-D LC 1-D GE ESI-MSMS or IPG strip C7250 from Carter et. al. The Plant Cell, 2004, 16, 3285–3303. 2D-On-line 2D-Off-line (3D ?) C7250 from Carter et. al. The Plant Cell, 2004, 16, 3285–3303. C7250 Depleted blood plasma (3500 – 9000 proteins ??) IEF (liq) 20 fractions 1. dimension LC (RP) 1600 fractions 2. dimension GE (1D/2D) “ ∞ “ fractions 3/4. dimension from H. Wang, Molecular & Cellular Proteomics, 2005, 4, 618–625. 0. dimension Example of multidimensional proteome analysis (screening) C7250 C7250 A draft map of the human proteome Min-Sik Kim et al., Nature 509, 575-581 doi:10.1038/nature13302 293 000 non redundant peptides corresponding to proteins encoded by 17294 genes from A.D. Zoumaro-Djayoon et al. / Methods 56 (2012) 268–274 Targeted separation - immunoaffinity fractionation (Y(phos)) C7250 from K. Bluemlein et al. / Nature protocols 6 (2011) 859 –869 Targeted MS analysis of selected proteins C7250 Parent mass Fragment mass CID Q1 Q3 Q2 Q1 „fix“ Q3 „fix“ * quadrupole Q1 and Q3 are fixed to selected values of m/z ( Q1-precursor and Q3- selected fragment), only precursors displaying production of selected fragment during fragmentation in collisional cell are recorded * enables to follow tens of reactions (transitions) during analytical run (MRM) BLESSED High throughput C7250 + Miniaturization - chip technology C7250 pic01454 Protein quantification by MS Functional Genomics and Proteomics National Centre for Biomolecular Research Faculty of Science Masaryk University Protein quantification by MS Approaches: using isotopically different tags * Absolute quantification determination of protein concentration (amount) by addition of corresponding standard with known amount (AQUA, PSAQ) * Relative quantification evaluation of relative changes of the protein content in compared samples label free metods of absolute and relative quantification based on statistical processing of MS, or MS/MS data advantage of this approach is possibility of comparison of unlimited number of samples and absence of derivatization reaction or isotopically labeled standards C7250 Relative quantification approaches Protein sample B Digestion of pooled samples Identical peptides from different samples are distinguished 100% Protein sample A SILAC, in-vivo LC-MS LC-MS/MS (iTRAQ) ICAT, ICPL, iTRAQ Separated digestion of samples iTRAQ, 16O/ 18O Identical peptides in MS mode are not distinguished MS/MS C7250 W. Yan, S.S. Chen, Briefings in functional genomics and proteomics 4 (1), 1–12, (2005) Overview of relative quantification methods C7250 Stable Isotope Labelling with Amino acids in Cell culture (SILAC) * in vivo * proteins are labeled by growing cells in media containing isotopically labeled amino acids (e.g. 2H-Leu, 13C-Lys, 13C-Tyr,13C-Arg, 13C/15N-Arg) A B AA labeled cell growing combining purification digestion MS quantification AA normal picture from Ong et al.: MCP 1(2002), 376 C7250 ICAT ... Isotope-Coded Affinity Tags technology for protein expression analysis ü improved quantitation of a wider range of proteins ü overcomes limitations of 2-D gel method (e.g. membrane, low abundant proteins) Ø tags specific for cysteine-containing peptides (reduction of sample complexity) Ø easy automation of a procedure C7250 icatfig2_z ICAT analysis peak ratios = relative abundance C7250 from Ong et al.: MCP 1 (2002), 376 Comparison of in vivo and in vitro quantification methods (SILAC vs ICAT) Cell cultures C7250 Mass Coded Abundance Tagging (MCAT) Cagney G., Emili A.: Nature Biotechnol 20 (2002), 163-170 * tryptic digestion * modification of digest of selected sample (K) * * * * * * * * * * * mixing nemodif/modif in ratio 1:1 D = 42 Da C7250 1:1 Cagney G., Emili A.: Nature Biotechnol 20 (2002), 163-170 MCAT C7250 MCAT Cagney G., Emili A.: Nature Biotechnol 20 (2002), 163-170 peak ratios = relative abundance C7250 MCAT possibility of utilization of derivatization for de novo sequencing b ions unchanged , y ions in doublets (42 Da) C7250 Kyanotrihydroboritan sodný Reductive alkylation - dimethylation - lysine and N-term of peptide - isotopically labeled formaldehyde (D, 13C) Hsu et al., Anal.Chem. 2003 C7250 http://pubs.rsc.org/services/images/RSCpubs.ePlatform.Service.FreeContent.ImageService.svc/ImageSer vice/Articleimage/2014/CC/c3cc47998f/c3cc47998f-f1_hi-res.gif Wu et al., Chem. Commun. 2014 C7250 Reductive alkylation - dimethylation Wu et al., Chem. Commun. 2014 C7250 Reductive alkylation - dimethylation iTRAQ * isobaric tags (4, 8), preferentially on Lys * labeled samples have the same behavior during LC separation and MS analysis * quantification based on intensity ratios of reporter ions after MS/MS 1:1:1:1 Applied Biosystems C7250 similarly TMT tags (Thermo Fisher Scientific) – 6/10 tags (see later) iTRAQ® Reagent-8Plex Protein Quantitation-figure1 iTRAQ Applied Biosystems C7250 Applied Biosystems iTRAQ C7250 Applied Biosystems iTRAQ C7250 TMT značky http://planetorbitrap.com/data/fe/image/Workflows_TMT_v3.jpg TMT labels Tandem Mass Tags from http://planetorbitrap.com C7250 TMT C7250 Thermo Fischer Scientific •isobaric labels (up to 16-plex) •MS/MS •Cysteine-reactive mass tags (6-plex) quantitation of the relative abundance of cysteine modifications, such as S- nitrosylation, oxidation and disulfide bonds •Carbonyl-reactive mass tags (6-plex) glycan, steroids, or oxidized proteins quantification lysine labeling TMT C7250 Procedure summary for parallel isobaric labeling for tandem mass spectrometry Thermo Fischer Scientific Analysis protocol summary for tandem mass spectrometry with 6-plex isobaric tags Relative quantitation by tandem mass spectrometry with TMT10plex Reagents Reagents contain different numbers and combinations of 13C and 15N isotopes in the mass reporter. The different isotopes result in a 10-plex set of tags that have mass differences in the reporter that can be detected using high resolution Orbitrap MS instruments. TMT C7250 Thermo Fischer Scientific EASI-tag Easily Abstractable Sulfoxide-based Isobaric tag bioRxiv preprint first posted online Nov. 27, 2017; doi: http://dx.doi.org/10.1101/225649 (a) Molecular structures of the triplex version of EASI-tag. The isobaric labeling reagents are composed in a modular way of four functional groups and feature a central sulfoxide moiety, which introduces an asymmetric, low-energy cleavage site (zig-zag lines indicate fragmentation site). The stable-isotope labeled positions of the neutral loss and equalizer group for multiplexing are indicated by asterisks. Standard labeling protocols can be applied to couple peptides via the amine-reactive moiety. (b) Mass spectra of an EASI-tag-labeled yeast peptide mixed in a ratio of 1:3:10. HCD fragmentation of the doubly charged precursor ion abstracts the neutral loss group and yields the peptide-coupled reporter ion cluster. C7250 EASI-tag Easily Abstractable Sulfoxide-based Isobaric tag C7250 (c) Co-isolation of the natural isotope cluster in a standard isolation window centered on the precursor ion (upper panel) convolutes the relative abundance of peptide coupled reporter ions. An asymmetric isolation window (lower panel) that suppresses the signal from adjacent isotope peaks and enables direct quantification of reporter ions. (d) The precursor mass information is retained in the peptide-coupled reporter ions for EASI-tag labeled peptides. Colored peaks indicate the peptide-coupled reporter ions from an identified yeast peptide in a two proteome experiment (mixing ratios: 1:3:10 for yeast & 1:1:1 for human). Grey peaks are peptide-coupled reporter ions from a co-isolated peptide. (e) EASI-tag- and TMT-labeled HeLa peptides were fragmented with normalized collision energies between 10 and 34. (N = 17,565 precursors for EASI-tag & 20,610 for TMT) bioRxiv preprint first posted online Nov. 27, 2017; doi: http://dx.doi.org/10.1101/225649 Label – free approach •No labels •Samples measured individually •Comparison of „unlimited“ number of samples Critical steps: •data normalization •imputation of missing values (DIA – reduction of number of missing values) LC-MS/MS noise removal peak detection intensity/area calculation for individual peaks normalization imputation of missing values calculation of fold changes for individual proteins among samples Statistics evaluation significant changes in proteome induced by stimuli under study C7250 Identification based on MS/MS data, connection of identity to individual signals (intensity, area) Label – free approach Violin graphs and number of observed values: violins width corresponds to the number of values not normalized 3582 3592 3503 3571 3534 3626 3567 35 30 25 20 3556 3552 Violin graphs and number of observed values: violins width corresponds to the number of values loessF normalized 3582 3592 3503 3571 3534 3626 3567 3556 3552 35 25 20 Density (dark blue->dark red) MA plot ('x-y' on matrix red curve is estimated nonparametric lowess model dashed line is unity line (x=y) —10 —10 10 5.0 2.5 0.0 —2.5 —5.0 —10 5.0 0 2.5 0.0 —2.5 —5.0 5.0 2.5 —1 0.0 —2.5 —5.0 —7.5 5.0 0 2.5 0.0 —2.5 —5.0 20 5-1 ID 20 5-2 ID 20 5-5 ID 20 5-6 ID 20 5-7 ID I essF normalized data 20 5-8 ID 20 6-2 ID 20 6-3 ID 20 6-4 ID 20 6-5 ID 20 6-8 ID MA plots - dependence of (x-y) on (x+y)/2 C7250 Label – free approach Volcano plot with selected categories visualization thresholds: logFC 1, P 0.01 Not sign. (8372) Down&Sign. (14) up&Sign. (22) Significant (54) LIMMA_KO-WT.logFC C7250 Flow_01 Ø AQUA Peptide Selection Ø Order selected peptide isotopically labeled (15N, 13C) Ø Adding labeled peptide to protein mix Ø Digest Ø Analyze by LC-MS/MS to quantitate protein of interest Select an optimal tryptic peptide and stable isotope amino acid from the sequence of your protein of interest Optimize LC-MS/MS separation protocol for quantitation Absolute quantification using AQUA peptides Only for selected protein C7250 Absolute quantification C7250 AQUA peptides QconCAT PSAQ synthesis of isotopically labeled proteotypic peptides construction of artificial gene for expression of proteotypic peptides originated from up to 20 proteins expression in E.coli using labeled medium purification of artificial protein MS analysis addition of known amount into sample digestion expression of the whole izotopically labeled protein (plus tag for purification) Rivers at al., MCP 6, 1416 (2007) protein purification Brun et al., MCP 6, 2139 (2007) MS analysis addition of known amount into sample digestion MS analysis addition of known amount into sample digestion Absolute quantification Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) N.L. Anderson, J. Proteome Res., 3, 235 (2004) MRM PRM S. Pan, J. Proteome Res., 8, 787 (2009) C7250 Targeted MS/MS analysis of selected proteins relative /absolute quantification multiple reaction monitoring (MRM) screening – selection of candidate protein method establishment (selection of MRM transitions – peptide + selected fragment) Protein mixture In-solution digestion Peptides LC-MS/MS – MRM mode final analysis and data processing http://www.srmatlas.org C7250 Similarly PRM Multiplex Immuno-Liquid Chromatography−Mass Spectrometry− Parallel Reaction Monitoring (LC−MS−PRM) Quantitation of immune markers CD8A, CD4, LAG3, PD1, PD-L1, and PD-L2 in Frozen Human Tissues C7250 Zhang et al., J. Proteome Res. 2018, 17, 3932−3940 Accurate MS-based Rab10 Phosphorylation Stoichiometry C7250 assay to measure increased phospho Rab levels using synthetic stable isotope-labeled analogues for both phosphorylated and non-phosphorylated tryptic peptides surrounding Rab10-Thr73 Limit of detection (LOD) of SIL Rab10-pThr73 tryptic peptide (FHpTITTSYYR) with various acquisition methods; full MS, SIM, mxSIM and PRM. Karayel et al., Mol Cell Proteomics (2020) 19(9) 1546–1560 Lawless C., MCP,15, 1309–1322, 2016. Absolute Quantification of over 1800 Yeast Proteins C7250 Golden_Road The end