Slide 1

Labeling and QuantitationCH 908: Mass Spectrometry Lecture 12Derivatization of Proteins/Peptides: PurposesSeparationDetection/AnalysisTo improve chromatographic properties:- Resolution;- RetainingAnalyte isolation/Sample clean-upTo improve sensitivity (UV, LIF, MS);To improve analyte stability;Differential analysis & QuantitationTo improve MS/MS sequencing (directed fragmentation)Multi-function tags combine 2 or more functions - are preferred!Labels widely used and commercially availableThe Molecular Probes Handbook A Guide to Fluorescent Probes and Labeling Technologies

LabelsLinkers

(all chemistries for various target molecules or objects)Classification of current quantitative proteomic techniques

Spiking with an isotopically labeled analogM.Miyagi , K.C. S. Rao Mass Spectrom. Rev. Vol.26, 1 Pages: 121-136Copyright 2007 Wiley Periodicals, Inc., A Wiley Company

Electrophoresis. 1997 Oct; 18(11): 2071-7.Unl M, Morgan ME, Minden JS.Difference gel electrophoresis: a single gel method for detecting changes in protein extracts.Differential Gel Electrophoresis(In-gel Quantitation)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 spectrometrybased proteomics turns quantitativeShao-En Ong & Matthias Mann Nature Chemical Biology , 252 - 262 (2005)Labeling with stable isotopesHigh 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)

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 51425149R = 0, ratioobs does not vary with time and always equals ratiotrue. R = 0.025, the deuterated peptide () would elute 1.5 s faster, and ratioobs varies continuously across the peak. 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 DTypes of labeling workflows in quantitative proteomics

Mass spectrometrybased proteomics turns quantitativeShao-En Ong & Matthias Mann Nature Chemical Biology , 252 - 262 (2005)Least prone to errorsUncompen-sated quantitation errors

Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MSScott A. Gerber, John Rush, Olaf Stemman, Marc W. Kirschner, and Steven P. GygiPNAS 2003;100:6940-6945Spiking with an internally labeled stable isotope standard: The AQUA strategy Absolute quantification of proteins and phosphoproteins using the AQUA strategy. The strategy has two stages. Stage 1 involves the selection and standard synthesis of a peptide (or phosphopeptide denoted by pS) from the protein of interest. During synthesis, stable isotopes are incorporated (e.g., 13C, 15N, etc.) at a single amino acid residue such as the leucine shown here denoted by *. These peptide internal standards are analyzed by MS/MS to examine peptide fragmentation patterns. The mass spectrometer is next set up to perform a SRM analysis in which a specific precursor-to-product ion transition is measured. Stage 2 is the implementation of the new peptide internal standard for precise quantification. Protein is harvested from a biological sample and proteolyzed with trypsin in the presence of the AQUA internal standard peptide/phosphopeptide. An LCSRM experiment then measures the abundance of a specific fragment ion from both the native peptide and the synthesized peptide as a function of reverse-phase chromatographic retention time. The absolute quantification is determined by comparing the abundance of the known AQUA internal standard peptide with the native peptide.Classification of current quantitative proteomic techniques

Spiking with an isotopically labeled analogM.Miyagi , K.C. S. Rao Mass Spectrom. Rev. Vol.26, 1 Pages: 121-136Copyright 2007 Wiley Periodicals, Inc., A Wiley CompanySILAC stands for Stable Isotope LAbeling in CultureStable 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 AAs)Isotopically labeled amino acids used: Leu D3 (allows differentiation between Leu and Ile), Met D3, Gly D2, Arg 13C6, Arg 13C615N4, etc.In-vivo metabolic labeling (SILAC)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 AdvantagesLimitationsUp to 100% labeling efficiency can be achievedThe label is introduced at the earliest possible stage of the analysis => minimal errorsAdditional sequence information from m: # of labeled amino acids can aid in identificationThe 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 ProteinsA combination of top-down approach with quantitationWhole proteins can be labeled with efficiency close to 100% (impossible to achieve by chemical labeling due to hindered access to AAs 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

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.(1)(2) (3)Classification of current quantitative proteomic techniques

Spiking with an isotopically labeled analogM.Miyagi , K.C. S. Rao Mass Spectrom. Rev. Vol.26, 1 Pages: 121-136Copyright 2007 Wiley Periodicals, Inc., A Wiley CompanyEnzymatic (18O) labelingThe proteolytic enzymes catalyze the following two reactions:

Proteases can catalyze incorporation of either 1 or 2 18O atoms

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 2733Enzymatic labeling: + and AdvantagesLimitationsApplicable to all types of biological samples; Effective with very low sample amounts (as low as 1-4 g of total protein, or 10,000 cells, in a recent report) No side reactions and byproducts, which are a general problem of chemical labeling. 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...Classification of current quantitative proteomic techniques

Spiking with an isotopically labeled analogM.Miyagi , K.C. S. Rao Mass Spectrom. Rev. Vol.26, 1 Pages: 121-136Copyright 2007 Wiley Periodicals, Inc., A Wiley CompanyChemical labelingLabel is an isotope-bearing molecule introduced by a chemical reactionMultifunctional labels can be designed

Julka, S.; Regnier, F. J. of Proteome Res. 2004, 3, 350-363Martin Mnchbach, Manfredo Quadroni, Giovanni Miotto, and Peter JamesQuantitation and Facilitated de Novo Sequencing of Proteins by Isotopic N-Terminal Labeling of Peptides with a Fragmentation-Directing Moiety Anal. Chem., 72 (17), 4047 -4057, 2000

ICAT (Isotope Coated Affinity Tags)1st generation

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.Reacts with SH-groups of CysFor affinity isolation of labeled peptides using an avidin LC columnIsotope label carrier25

ICAT analysis flowchart

Gygi, S. P.; Aebersold, R. Curr. Opin. Chem. Biol. 2000, 4, 489-94.ICAT: + and AdvantagesLimitationsMultipurpose!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 BiosystemsCleavable ICAT reagentAdvantages 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 reagentsThe 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 121200 fmol BSA on column (assuming 100% efficiency through cICAT procedures).B The recovery and precision were measured with the same fetal bovine serum sample (0.1g 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 80C 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 50g/mL BSA.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 45434552Total amount of protein used in various cICAT studies ranged from 4.4 mg to 200 g.

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

Targeting phosphopeptides: PhIATTags for MS/MS analysis: iTRAQA 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.Isobaric tags (iTRAQ)

Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents Ross PL, ... Pappin D.J.(2004) Mol Cell Proteomics 3(12): 1154-69iTRAQ (continued)

iTRAQ for quantitation

Reporter ion region in a MS/MS spectrumA tag that influences MS signal and MS/MS fragmentationN-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

O

Conversion of lysine to homoarginine

Derivatizing agent - O-methylisourea;Increase signal intensities of Lys-containing peptides in MALDITOFMS;Conversion efficiency for -amino groups of Lys is 100%, side reactions are minimal, and the reagents are inexpensive and readily available

Commercialized as Lys-Tag 4H (Agilent)Mass defect labelingMass Difference from Nucleon Value of the Most Abundant Isotope of the Elements Found in ProteinsElementmass defect (amu) 12C01H 0.007816O 0.005115N 0.003132S 0.0279Mass 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): 34173423 Typical MM distribution for tryptic peptides

Histogram of the molecular mass distribution of the predicted tryptic peptides of M. maripaludis over the range 15001503 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): 34173423Cys-alkylation reaction (to introduce mass defect label)

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): 34173423MDL (Mass Deficit Label) reagent - 2,4-dibromo-(2-iodo)acetanilide 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]41Additional 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 spectrometrybased proteomics turns quantitativeShao-En Ong & Matthias Mann Nature Chemical Biology , 252 - 262 (2005)Classification of current quantitative proteomic techniques

Spiking with an isotopically labeled analogWe are still here!MS/MS tags: directed fragmentationWhy 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

Sequence: FGQGEAAPVVAPAPAPAPEVQTKMASCOT score 252MS/MS: The GoodAn Example of a Good Spectrum

MS/MS: The Bad OK SpectrumQNNFNAVRMascot Score 43MS/MS: The Ugly Poor fragmentation

Sequence: GALSAVVADSR, MASCOT score 10Charge-directed fragmentationFixed-charge N-terminal tags: quaternary ammonium or phosphonium cationsFirst offered for sector tandem MS instruments (Biemann et.al.) in early 1990ies

49Charge-directed fragmentation of -thymosinZaia, J.; Biemann, K., Comparison of charged derivatives for high energy collision-induced dissociation tandem mass spectrometry Anal. Chem. 1995, 6, 428-436

-thymosin

Charge-directed fragmentation (more 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 MALDI-TOF/TOF (high-energy collisions)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 2085209670.0387.4704.81022.21339.61657.0Mass (m/z)6.2E+40102030405060708090100% Intensityy9(+1)y14(+1)y6(+1)y1(+1)y7(+1)y9-17(+1)y13(+1)y10(+1)y2(+1)y8(+1)y4(+1)y3(+1)b7-18(+1)y5(+1)Ry11(+1)y2 - 17(+1)b14(+1)EGVNDNEEGFFSAR NativeA3.7E+470.0508.2946.41384.61822.82261.0Mass (m/z)0102030405060708090100% Intensity b*(+1)1986.8705559.2617b5(+1)a1(+1)b1(+1)b4(+1)b13(+1)527.2357b8(+1)a7(+1)b6(+1)a10(+1)a11(+1)77.2654EGVNDNEEGFFSAR TMPP tagBNo sequence fragments!

A Mobile Proton Theory of Peptide FragmentationThe most stable protonated form may not be the fragmenting structureFragmentation (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 spectrumDongre, A. R.; Jones, J. L.; Somogyi, A.; Wysocki, V. H., J. Am. Chem. Soc. 1996, 118, 8365-8374.

Order of basicity:Guanidine group of Arg > N-term N > Carbonyl O > Amide N => a dynamic equilibrium of structuresLocalization of a Proton on a Protonated Peptide

The Fragmenting Structure of a Protonated PeptideThe most stable protonated form is NOT the fragmenting structure!Sulfonated peptide: adding 1 extra mobile proton

Tags containing a sulfo-group OSuOONHO3SMeOHHAlexa Fluor 350 succinimide ester Added mass 295.01

(Molecular Probes)Keough, T.; Youngquist, R. S.; Lacey, M. P., Anal. Chem. 2003, 75, 156A-165A57Labeling with Alexa Fluor 350

Sequence: GALSAVVADSR, MASCOT score 10

Mascot Score 95Monoisotopic mass of neutral peptide (Mr): 1339.571Fixed 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 Monoisotopic mass of neutral peptide (Mr): 1044.556 Ions Score: 10 Matches (Bold Red): 5/63 fragment ions using 7 most intense peaks Low MS signal intensity precursorSVDEAANSDIVDK1546.01588.21630.41672.61714.81757.0Mass (m/z)2.4E+40102030405060708090100% Intensity1570.67721552.67201719.51541699.67681604.70001669.60001627.66001565.7336

MS: S/N 9, 350 counts MS/MS: Mascot score 119High quality spectrum obtained from a MS precursor with S/N< 10(E.coli protein digest labeled with Alexa Fluor 350)Complimentary information?PeptidesProteins147common(43.6%)105 unique(31.2%)NativeAlexa Tagged85 unique(25.2%)124common(14.5%)295 unique(34.6%)433 unique(50.8%)NativeAlexa TaggedLC-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.Hydrophilic, Lys-peptidesHydrophobic, Arg-peptides