1. CH 908: Mass Spectrometry Lecture 12

2. Derivatization of Proteins/Peptides: Purposes Separation Detection/Analysis To improve chromatographic properties: - Resolution; - Retaining Analyte isolation/ Sample clean-up To improve sensitivity (UV, LIF, MS); To improve analyte stability; Differential analysis & Quantitation To improve MS/MS sequencing (directed fragmentation) Multi-function tags – combine 2 or more functions - are preferred!

3. 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)

4. 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

5. 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. Differential Gel Electrophoresis (In-gel Quantitation)

6. 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;

7. 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)

8. 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 13 C, 15 N, 18 O are preferred to D (less isotopic separation)

9. 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 5142–5149 (A)R = 0, ratio obs does not vary with time and always equals ratio true. (B)R = 0.025, the deuterated peptide (·) would elute 1.5 s faster, and ratio obs varies continuously across the peak. (C)R = 0.5, the deuterated peptide (·) would elute 30 s faster than the nondeuterated peptide (▪), and ratio obs varies continuously across the peak and is very high H/D-labeled pairs may separate as much as 1 min in RP HPLC => 13 C, 15 N, 18 O are preferred to D

10. Types of labeling workflows in quantitative proteomics Mass spectrometry–based proteomics turns quantitative Shao-En Ong & Matthias Mann Nature Chemical Biology, 252 - 262 (2005) Least prone to errors Uncompen- sated quantitation errors

11. 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 Spiking with an internally labeled stable isotope standard: The AQUA strategy

12. 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

13. SILAC stands for Stable Isotope LAbeling in Culture Stable isotopes, such as 15 N or 13 C, 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 D 3 (allows differentiation between Leu and Ile), Met D 3, Gly D 2, Arg 13 C 6, Arg 13 C 6 15 N 4, 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)

14. SILAC scheme Mumby and Brekken Genome Biology 2005 6:230

15. 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

16. 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.

17. 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)

18. 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

19. Enzymatic ( 18 O) labeling The proteolytic enzymes catalyze the following two reactions: Proteases can catalyze incorporation of either 1 or 2 18 O atoms

20. 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 18 O 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

21. Enzymatic labeling: + and – Advantages Limitations 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. Small ∆m (2 or 4) => isotopic envelope overlap; Incomplete incorporation of 2 nd 18 O 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 18 H 2 O...

22. 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

23. 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

24. 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 Fragmentation- Directing Moiety Anal. Chem., 72 (17), 4047 -4057, 2000

25. ICAT (Isotope Coated Affinity Tags) 1 st 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 Cys For affinity isolation of labeled peptides using an avidin LC column Isotope label carrier

26. ICAT analysis flowchart Gygi, S. P.; Aebersold, R. Curr. Opin. Chem. Biol. 2000, 4, 489-94.

27. 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)

28. New generation of ICAT : a cleavable reagent Applied Biosystems

29. Cleavable ICAT reagent Advantages compared to the old ICAT reagent 13 C 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.

30. 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. 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 Total amount of protein used in various cICAT studies ranged from 4.4 mg to 200 µg.

31. 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: PhIAT

32. 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.

33. 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): 1154-69

34. iTRAQ (continued)

35. iTRAQ for quantitation Reporter ion region in a MS/MS spectrum

36. A tag that influences MS signal and MS/MS fragmentation 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 O

37. Conversion of lysine to homoarginine Derivatizing agent - O-methylisourea; Increase signal intensities of Lys- containing peptides in MALDI–TOF MS; Conversion efficiency for ε-amino groups of Lys is 100%, side reactions are minimal, and the reagents are inexpensive and readily available Commercialized as Lys-Tag 4H (Agilent)

38. Mass defect labeling Mass Difference from Nucleon Value of the Most Abundant Isotope of the Elements Found in Proteins Elementmass defect (amu) 12 C0 1H1H0.0078 16 O−0.0051 15 N0.0031 32 S−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

39. 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

40. Cys-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): 3417–3423 MDL (Mass Deficit Label) reagent - 2,4-dibromo-(2 ′ -iodo)acetanilide

41. MS of peptides labeled with MDL The reagent was tested on a 15 N-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]

42. 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.

43. 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)

44. Classification of current quantitative proteomic techniques Spiking with an isotopically labeled analog We are still here!

45. 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

46. Sequence: FGQGEAAPVVAPAPAPAPEVQTK MASCOT score 252 MS/MS: The Good… An Example of a Good Spectrum

47. MS/MS: The Bad… “OK” Spectrum QNNFNAVR Mascot Score 43

48. MS/MS: The Ugly Poor fragmentation Sequence: GALSAVVADSR, MASCOT score 10

49. Charge-directed fragmentation Fixed-charge N- terminal tags: quaternary ammonium or phosphonium cations First offered for sector tandem MS instruments (Biemann et.al.) in early 1990ies

50. Charge-directed fragmentation of 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 α-thymosin

51. 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

52. 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 2085–2096 No sequence fragments!

53. 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.

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

55. The Fragmenting Structure of a Protonated Peptide The most stable protonated form is NOT the fragmenting structure!

56. Sulfonated peptide: adding 1 extra mobile proton

57. Tags containing a sulfo-group OSu O O N H O 3 S Me O H H Alexa Fluor 350 succinimide ester Added mass 295.01 (Molecular Probes) Keough, T.; Youngquist, R. S.; Lacey, M. P., Anal. Chem. 2003, 75, 156A-165A

58. Labeling with Alexa Fluor 350 Sequence: GALSAVVADSR, MASCOT score 10 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 Monoisotopic mass of neutral peptide (Mr): 1044.556 Ions Score: 10 Matches (Bold Red): 5/63 fragment ions using 7 most intense peaks

59. Low MS signal intensity precursor High quality spectrum obtained from a MS precursor with S/N< 10 (E.coli protein digest labeled with Alexa Fluor 350)

60. Complimentary information? PeptidesProteins 147 common (43.6%) 105 unique (31.2%) NativeAlexa Tagged 85 unique (25.2%) 124 common (14.5%) 295 unique (34.6%) 433 unique (50.8%) NativeAlexa Tagged 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. Hydrophilic, Lys- peptides Hydrophobic, Arg- peptides