Chemical tricks which aid MS of proteins… CH 908: Mass Spectrometry Lecture 12 Objectives • An overview of the chemical labelling concepts currently being explored. – Labelling for quantitation – Labelling for separation/isolation of specific sub-fractions (for example, phosphoproteins) – Labelling the surface of a protein for structural characterization. – Labelling to make the mass spectrometry and MSMS data better (or worse). – Metabolic labelling for study of cellular processes. – isobaric labels – crosslinking studies – … Derivatization of Proteins/Peptides: Purposes Separation Detection/Analysis • To improve chromatographic properties: - Resolution; - Retaining • To improve sensitivity (UV, LIF, MS); • Analyte isolation/ Sample clean-up • To improve MS/MS sequencing (directed fragmentation) • To improve analyte stability; • Differential analysis & Quantitation Multi-function tags – combine 2 or more functions - are preferred! Labels – widely used and commercially available The Molecular Probes Handbook — A Guide to Fluorescent Probes and Labeling Technologies Labels Linkers (all chemistries for various target molecules or objects) Classification of current quantitative proteomic techniques Spiking with an isotopically labeled analog M.Miyagi , K.C. S. Rao Mass Spectrom. Rev. Vol.26, 1 Pages: 121-136 Copyright © 2007 Wiley Periodicals, Inc., A Wiley Company Differential Gel Electrophoresis (In-gel Quantitation) Electrophoresis. 1997 Oct; 18(11): 2071-7. Unlü M, Morgan ME, Minden JS. Difference gel electrophoresis: a single gel method for detecting changes in protein extracts. Isotope dilution mass spectrometry (IDMS) • IDMS is the use of an enriched isotope of the element of interest as the internal standard; • Has been known for nearly 50 years; • The IDMS technique involves the addition of a known amount of an enriched isotope of the element of interest to the sample. - made prior to sample preparation - the sample concentration can be calculated by measuring the isotope ratio of the sample and sample + spike; • Not applicable to monoisotopic elements • First work cited for peptides: Desiderio DM, Kai M. Preparation of stable isotope-incorporated peptide internal standards for field desorption mass spectrometry quantification of peptides in biologic tissue. Biomed Mass Spectrom. 1983 Aug;10(8):471-9; Labeling with stable isotopes: MS and LC views Mass spectrometry–based proteomics turns quantitative Shao-En Ong & Matthias Mann Nature Chemical Biology , 252 - 262 (2005) Labeling with stable isotopes • High labeling efficiency (ideally 100%) is a key for successful quantitation • The earlier the label is introduced – the lesser uncompensated quantitation errors • 13C, 15N, 18O are preferred to D (less isotopic separation) Isotopic separation (H/D) (A) R = 0, ratioobs does not vary with time and always equals ratiotrue. (B) R = 0.025, the deuterated peptide (·) would elute 1.5 s faster, and ratioobs varies continuously across the peak. (C) R = 0.5, the deuterated peptide (·) would elute 30 s faster than the nondeuterated peptide (▪), and ratioobs varies continuously across the peak and is very high H/D-labeled pairs may separate as much as 1 min in RP HPLC => 13C, 15N, 18O are preferred to D Fractionation of Isotopically Labeled Peptides in Quantitative Proteomics. R. Zhang, C. S. Sioma, S. Wang, and F. E. Regnier Anal. Chem., 2001, 73 (21), pp 5142–5149 Types of labeling workflows in quantitative proteomics Least prone to errors Uncompensated quantitation errors Mass spectrometry–based proteomics turns quantitative Shao-En Ong & Matthias Mann Nature Chemical Biology , 252 - 262 (2005) Spiking with an internally labeled stable isotope standard: The AQUA strategy Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS Scott A. Gerber, John Rush, Olaf Stemman, Marc W. Kirschner, and Steven P. Gygi PNAS 2003;100:6940-6945 Classification of current quantitative proteomic techniques Spiking with an isotopically labeled analog M.Miyagi , K.C. S. Rao Mass Spectrom. Rev. Vol.26, 1 Pages: 121-136 Copyright © 2007 Wiley Periodicals, Inc., A Wiley Company In-vivo metabolic labeling (SILAC) • SILAC stands for Stable Isotope LAbeling in Culture • Stable isotopes, such as 15N or 13C, can be incorporated into proteins during normal biosynthesis by growing cells in media supplemented with stable-isotope containing nutrients (typically AA’s) • Isotopically labeled amino acids used: Leu D3 (allows differentiation between Leu and Ile), Met D3, Gly D2, Arg 13C , Arg 13C 15N , etc. 6 6 4 A practical recipe for stable isotope labeling by amino acids in cell culture (SILAC) Shao-En Ong and Matthias Mann Nature Protocols , 2650 - 2660 (2007) SILAC scheme Mumby and Brekken Genome Biology 2005 6:230 SILAC: + and – Advantages Limitations • Up to 100% labeling efficiency can be achieved • The label is introduced at the earliest possible stage of the analysis => minimal errors • Additional sequence information from ∆m: # of labeled amino acids can aid in identification • The only isotopic labeling method for top-down protein quantitation • The main limitation: SILAC is applicable only for cells which can be cultivated; • Mass window between the two isotopic versions of peptides is not uniform, and the data processing tools for this are not readily available Top-Down Quantitation and Characterization of SILAC-Labeled Proteins • A combination of top-down approach with quantitation • Whole proteins can be labeled with efficiency close to 100% (impossible to achieve by chemical labeling due to hindered access to AA’s in internal regions); • Complete labeling is a precondition for successful quantitative top-down analysis J Am Soc Mass Spectrom. 2007 Nov;18(11):2058-64. Top-down quantitation and characterization of SILAC-labeled proteins. Waanders LF, Hanke S, Mann M. Top-Down Quantitation and Characterization of SILAC-Labeled Proteins (1) (2) (3) The effects of incomplete isotope enrichment and incomplete mass labeling are modeled, based on the 34+ peak of a theoretical protein of 55 kDa with average amino acid composition, labeled with heavy Arg and measured with 60,000 resolving power. (1) A 98% isotope enrichment of 13-C and 15-N in the heavy amino acids results in a shift of the total heavy isotopic cluster from 100% enrichment. The width and the intensity of the isotopic cluster remain unchanged. (2) In contrast, if only 98% of the arginines and lysines are labeled with heavy amino acids, the result is a significant spread of the signal. (3) With 95% labeling the signal is even more spread and the total intensity is reduced to 33% of the original signal. quantitative proteomic techniques Spiking with an isotopically labeled analog M.Miyagi , K.C. S. Rao Mass Spectrom. Rev. Vol.26, 1 Pages: 121-136 Copyright © 2007 Wiley Periodicals, Inc., A Wiley Company Enzymatic (18O) labeling • The proteolytic enzymes catalyze the following two reactions: Enzymatic labeling example Partial spectrum of the labeled and unlabeled dimer of HSP peptide CLNRQLpSSGVSE. Inset shows theoretical distribution of molecular ion envelope for the unlabeled dimer. Proteolytic 18O Labeling for Comparative Proteomics: Evaluation of Endoprotease Glu-C as the Catalytic Agent K. J. Reynolds, X. Yao, and C. Fenselau, Journal of Proteome Research, 2002, 1 (1), pp 27–33 Enzymatic labeling: + and – Advantages • Applicable to all types of biological samples; • Effective with very low sample amounts (as low as 1-4 μg of total protein, or 10,000 cells, in a recent report) • No side reactions and byproducts, which are a general problem of chemical labeling. Limitations • Small ∆m (2 or 4) => isotopic envelope overlap; • Incomplete incorporation of 2nd 18O atom, the reaction is hard to control, and the rate of exchange differs with peptide size, type of amino acid, between enzymes and with peptide sequence; • C-terminal peptides are not labeled => singlets in MS => can be interpreted as having arisen from large changes in expression; • Losses during sample prep (dry out H2O, then reconstitute in 18H2O... quantitative proteomic techniques Spiking with an isotopically labeled analog M.Miyagi , K.C. S. Rao Mass Spectrom. Rev. Vol.26, 1 Pages: 121-136 Copyright © 2007 Wiley Periodicals, Inc., A Wiley Company Chemical labeling • Label is an isotope-bearing molecule introduced by a chemical reaction • Multifunctional labels can be designed Julka, S.; Regnier, F. J. of Proteome Res. 2004, 3, 350-363 Martin Münchbach, Manfredo Quadroni, Giovanni Miotto, and Peter James Quantitation and Facilitated de Novo Sequencing of Proteins by Isotopic N-Terminal Labeling of Peptides with a FragmentationDirecting Moiety Anal. Chem., 72 (17), 4047 -4057, 2000 ICAT (Isotope Coated Affinity Tags) 1st generation Reacts with SH-groups of Cys For affinity isolation of labeled peptides using an avidin LC column Isotope label carrier Gygi, S. P., B. Rist, et al. (1999). "Quantitative analysis of complex protein mixtures using isotope-coded affinity tags." Nat Biotechnol 17(10): 994-9. ICAT analysis flowchart Gygi, S. P.; Aebersold, R. Curr. Opin. Chem. Biol. 2000, 4, 489-94. ICAT: + and – Advantages Limitations • • • • • Multipurpose! Labeling protocol – alkylation (well-developed); Affinity isolation: simplified peptide mixtures; presence and # of Cys aids identification (constraint) The only commercially available method selective for a specific residue (Cys); enrichment allows proteomic studies on a much wider dynamic range than the other methods • • • H/D labeled peptides did not coelute during RP HPLC => less accurate quantitation; Bulky biotin molecule (m~500) shifted the masses of larger peptides outside the optimal mass range and also impaired the MS/MS CID spectra of the peptides; For peptides containing 2 Cys, ∆m (2 x 8= 16) overlapped with the same oxidized peptide (∆M = 16); Not suitable for proteins which do not contain Cys (e.g., ~8% of the yeast proteome) New generation of ICAT : a cleavable reagent Applied Biosystems Cleavable ICAT reagent Advantages compared to the old ICAT reagent • 13C instead of D => co-elution of ICAT labeled pairs from RP HPLC => improved quantification; • Acid-cleavable site in the reagent => removal of the biotin portion of the ICAT reagent tag prior to MS and MS/MS analysis; • Reduced tag fragmentation => improved quality of MS/MS data; • ∆m of 9 Daltons avoids possible confusion between oxidized methionine • and two cysteines labeled with ICAT reagents . The number of proteins detected with this second-generation cleavable reagent was larger than with the first generation reagent Investigation of the Linearity, Recovery and Precision of LC/Triple-Quad MRM-MS/MS with Six cICAT-Peptides Derived from BSA A The linearity for BSA quantification was calculated independently for the six cICAT-peptides over the range of 12–1200 fmol BSA on column (assuming 100% efficiency through cICAT procedures). B The recovery and precision were measured with the same fetal bovine serum sample (0.1μg total proteins); the recoveries were determined in triplicate, by spiking respectively at the level of 48 and 480 fmol BSA (on column) into samples; to determine precision, aliquots of the serum sample stored at −80°C were injected twice on two different days (n=4). C Detection limits of both systems were defined as the BSA amount on column (assuming 100% efficiency through cICAT procedures) that gave a S/N of 3; the conditions for quantitative LC/MS/MS were optimized using cICAT-peptides derived from 50μg/mL BSA. Total amount of protein used in various cICAT studies ranged from 4.4 mg to 200 µg. Utility of Cleavable Isotope-Coded Affinity-Tagged Reagents for Quantification of Low-Copy Proteins Induced by Methylprednisolone Using LC/ MS/MS J. Qu, W. J. Jusko, R. M. Straubinger Anal. Chem., 2006, 78 (13), pp 4543–4552 Targeting phosphopeptides: PhIAT Phosphoprotein Isotope-Coded Affinity Tag Approach for Isolating and Quantitating Phosphopeptides in Proteome-Wide Analyses Michael B. Goshe, Thomas P. Conrads, Ellen A. Panisko, Nicolas H. Angell, Timothy D. Veenstra, and Richard D. Smith Anal. Chem. 73 (11), 2578 -2586, 2001 Tags for MS/MS analysis: iTRAQ • A complimentary method to ICAT rather than an alternative to it; • Targets N-termini of peptides as an N-hydroxysuccinimide (NHS-) ester; • 4 isotopic variants, yet the tag is ISOBARIC in the MS mode; the difference shows up only after fragmentation of the tag in MS/MS mode - 4 samples can be run simultaneously; - single precursor selection. (iTRAQ) Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents Ross PL, ... Pappin D.J.(2004) Mol Cell Proteomics 3(12): 1154-69 iTRAQ (continued) iTRAQ for quantitation Reporter ion region in a MS/MS spectrum A tag that influences MS signal and MS/MS fragmentation O • • • • • • N-methylpiperazine (and similar structures, such as morpholine) is a weak base => additional protonation site => N-terminal labeling with basic tags leads to enhancement of peptide signals in the MS mode and also facilitates peptide fragmentation => More abundant b-ion series and decreased number of less informative internal cleavage fragments => More sequence ladder fragments and improved MS/MS spectra assignment => Higher confidence in protein ID Conversion of lysine to homoarginine • Derivatizing agent - Omethylisourea; • Increase signal intensities of Lys-containing peptides in MALDI–TOF MS; • Conversion efficiency for εamino groups of Lys is 100%, side reactions are minimal, and the reagents are inexpensive and readily available Mass defect labeling • Mass Difference from Nucleon Value of the Most Abundant Isotope of the Elements Found in Proteins Eleme nt mass defect (amu) 12C 0 1H 0.0078 16O −0.0051 15N 0.0031 32S −0.0279 Mass Defect Labeling of Cysteine for Improving Peptide Assignment in Shotgun Proteomic Analyses. H. Hernandez, S.Niehauser, S.A. Boltz, V. Gawandi, R. S. Phillips, and I. J. Amster, Anal Chem. 2006 May 15; 78(10): 3417–3423 Typical MM distribution for tryptic peptides Histogram of the molecular mass distribution of the predicted tryptic peptides of M. maripaludis over the range 1500–1503 Da, Illustrating the distribution of mass defects of peptides. The bin size is 0.01 amu. Peptide masses are observed to cluster in approximately one-third of the available mass space. Mass Defect Labeling of Cysteine for Improving Peptide Assignment in Shotgun Proteomic Analyses. H. Hernandez, S.Niehauser, S.A. Boltz, V. Gawandi, R. S. Phillips, and I. J. Amster, Anal Chem. 2006 May 15; 78(10): 3417–3423 Cys-alkylation reaction (to introduce mass defect label) MDL (Mass Deficit Label) reagent 2,4-dibromo-(2′-iodo)acetanilide Mass Defect Labeling of Cysteine for Improving Peptide Assignment in Shotgun Proteomic Analyses. H. Hernandez, S.Niehauser, S.A. Boltz, V. Gawandi, R. S. Phillips, and I. J. Amster, Anal Chem. 2006 May 15; 78(10): 3417–3423 MS of peptides labeled with MDL The reagent was tested on a 15N-metabolically labeled proteome from M. maripaludis. Proteins were identified by their accurate mass values and from their nitrogen stoichiometry. A total of 47% of the labeled peptides are identified versus 27% for the unlabeled peptides. [the same reference] Additional constraint: Phosphopeptides Distinctively large mass defect of phosphorus relative to H, C, and O (~0.3 Da) has the net result of off-setting the average mass of phosphopeptides to slightly lower mass than unmodified peptides of the same nominal molecular weight, often marking a peptide as phosphorylated simply on the basis of its mass. Stoichiometry of protein complexes • In a coprecipitation experiment, the goal is to identify proteins that bind differentially to wild-type and mutant baits (bait = studied protein + affinity tag). • Proteins that bind specifically to the bait or secondary interactors will give a ratio indicative of increased binding and enrichment. • Proteins that bind unspecifically to the affinity support or beads will show ratios similar to the mixing ratio. • Repeating the experiment with switched labels should result in inverse ratios, further increasing the specificity of the assay. Mass spectrometry–based proteomics turns quantitative Shao-En Ong & Matthias Mann Nature Chemical Biology , 252 - 262 (2005) Classification of current quantitative proteomic techniques Spiking with an isotopically labeled analog We are still here! MS/MS tags: directed fragmentation • Why do we need that? - De novo (old times); - Improve quality of the MS/MS spectra • Less than 20% of all peaks in MALDI-TOF MS spectra and < 5% of ESI-IT yield interpretable MS/MS spectra which result in peptide identification! • MS/MS data acquisition takes ~80% of MS work time MS/MS: The Good… An Example of a Good Spectrum Sequence: FGQGEAAPVVAPAPAPAPEVQTK MASCOT score 252 MS/MS: The Bad… “OK” Spectrum QNNFNAVR Mascot Score 43 MS/MS: The Ugly Poor fragmentation Sequence: GALSAVVADSR, MASCOT score 10 Charge-directed fragmentation • Fixed-charge Nterminal tags: quaternary ammonium or phosphonium cations • First offered for sector tandem MS instruments (Biemann et.al.) in early 1990ies thymosin α-thymosin Zaia, J.; Biemann, K., Comparison of charged derivatives for high energy collision-induced dissociation tandem mass spectrometry Anal. Chem. 1995, 6, 428-436 spectra) Zaia, J.; Biemann, K., Comparison of charged derivatives for high energy collision-induced dissociation tandem mass spectrometry Anal. Chem. 1995, 6, 428-436 Cationic tag labeling for MALDITOF/TOF (“high-energy” collisions) A 6.2E+4 % Intensity y9(+1) 100 EGVNDNEEGFFSAR 90 80 Native 70 60 y14(+1) 50 y6(+1) 40 y1(+1) 30 y7(+1) y9-17(+1) 20 R y2 - 17(+1) y10(+1) 10 y2(+1)y3(+1) y4(+1) y5(+1)b7-18(+1) y8(+1) y11(+1) y13(+1)b14(+1) 0 387.4 704.8 Mass (m/z) 1022.2 1339.6 1657.0 70.0 B 3.7E+4 b*(+1) EGVNDNEEGFFSAR TMPP tag % Intensity 100 90 80 70 60 50 40 30 20 10 77.2654 0 70.0 559.2617 a1(+1) 527.2357 b1(+1) 508.2 No sequence fragments! 1986.8705 b5(+1) b4(+1) b6(+1) a7(+1)b8(+1)a10(+1)a11(+1)b13(+1) 946.4 1384.6 Mass (m/z) 1822.8 2261.0 Coumarin Tags for Analysis of Peptides by MALDI-TOF MS and MS/MS. 2. Alexa Fluor 350 Tag for Increased Peptide and Protein Identification by LC-MALDI-TOF/TOF MS A.Pashkova, (...) E. Moskovets, and B. L. Karger Anal. Chem., 2005, 77 (7), pp 2085–2096 A Mobile Proton Theory of Peptide Fragmentation • The most stable protonated form may not be the fragmenting structure • Fragmentation (backbone) occurs due to the weakening of the amide bond, i.e. decrease of the bond order • Calculations showed that this will happen in the case of the protonation of the amide N • The more “mobile” (not localised) the proton, the more fragments in a MS/MS spectrum =>the more information from the spectrum Dongre, A. R.; Jones, J. L.; Somogyi, A.; Wysocki, V. H., J. Am. Chem. Soc. 1996, 118, 8365-8374. Localization of a Proton on a Protonated Peptide + H Order of basicity: Guanidine group of Arg > N-term N > Carbonyl O > Amide N NH2 HN => a dynamic equilibrium of structures + + H H R1 O + H O H N H2N NH R2 N H OH O The Fragmenting Structure of a Protonated Peptide The most stable protonated form is NOT the fragmenting structure! NH2 HN + NH H R1 O H N H2N O R2 N H OH O Sulfonated peptide: adding 1 extra mobile proton + NH2 H2N + NH H O O S O R1 O O H N N O R2 N H OH O Tags containing a sulfo-group H H N O O Alexa Fluor 350 succinimide ester O Added mass 295.01 O Su HO3S (Molecular Probes) Me O HO3S 3-sulfopropionic acid succinimide ester (CAF reagent) O Su Added mass 135.62 Keough, T.; Youngquist, R. S.; Lacey, M. P., Anal. Chem. 2003, 75, 156A-165A Labeling with Alexa Fluor 350 Sequence: GALSAVVADSR, MASCOT score 10 Monoisotopic mass of neutral peptide (Mr): 1044.556 Ions Score: 10 Matches (Bold Red): 5/63 fragment ions using 7 most intense peaks Mascot Score 95 Monoisotopic mass of neutral peptide (Mr): 1339.571 Fixed modifications: Guanidination (K) Variable modifications: N-term : Alexa (N-term) Ions Score: 95 Matches (Bold Red): 10/63 fragment ions using 11 most intense peaks Low MS signal intensity precursor SVDEAANSDIVDK 1570.6772 MS/MS: Mascot score 119 100 90 2.4E+4 80 % Intensity 70 60 50 40 MS: S/N 9, 350 counts 0 1546.0 1588.2 1630.4 1672.6 1719.5154 1699.6768 1669.6000 1627.6600 1604.7000 10 1565.7336 20 1552.6720 30 1714.8 1757.0 Mass (m/z) High quality spectrum obtained from a MS precursor with S/N< 10 (E.coli protein digest labeled with Alexa Fluor 350) information? Peptides Native 295 unique (34.6%) Proteins Alexa Tagged 124 common (14.5%) Hydrophobic, Argpeptides 433 unique (50.8%) Native 85 unique (25.2%) Alexa Tagged 147 common (43.6%) 105 unique (31.2%) Hydrophilic, Lyspeptides LC-MALDI-TOF/TOF MS analysis of tryptic peptides from the SCX fractions of an E. coli lysate revealed improved peptide scores, a doubling of the total number of peptides, and a 30% increase in the number of proteins identified, as a result of labeling with Alexa Fluor 350. Self Assessment • What good is an isobaric tag? Why does it work? • Why would you want to modify a protein to add a sulphate group? • What’s ICAT? What kinds of peptides does it work for? • What’s ITRAQ? What’s special about this tag? • Why would you want to isotopically label proteins or peptides?