1. Mass Spectrometry-Based Methods for Protein Identification Joseph A. Loo Department of Biological Chemistry David Geffen School of Medicine Department of Chemistry and Biochemistry University of California Los Angeles, CA USA

2. Genomics and Proteomics Characterizing many genes and gene products simultaneously

3. Proteomics Aids Biological Research Biology protein separation complex protein mixture mass spectrometry protein identification protein modification protein abundance

4. Proteomics - What is it? An assay to systematically analyze the diverse properties of proteins Biological processes are dynamic A quantitative comparison of states is required The study of protein expression and function on a genome scale n Purpose: Examine altered gene expression pathways in disease states and under different environmental conditions The completion of the human genome has provided researchers with the blueprint for life, and proteomics offers scientists the means for analyzing the expressed genome.

5. Genome to Proteome MTDLKASSLRALKLMDLTTLNDDDTDEKVIALCHQAKTPVGNTAAICIYP 51 RFIPIARKTLKEQGTPEIRIATVTNFPHGNDDIDIALAETRAAIAYGADE 101 VDVVFPYRALMAGNEQVGFDLVKACKEACAAANVLLKVIIETGELKDEAL 151 IRKASEISI Transcription dsDNA mRNA Translation Protein H2NH2NH2NH2NCOOH (Gene) The completion of the human genome has provided researchers with the blueprint for life, and proteomics offers scientists the means for analyzing the expressed genome. Mass spectrometry

6. Approaches for Protein Identification What is this protein? Molecular weight Isoelectric point Amino acid composition Other physical/chemical characteristics Partial or complete amino acid sequence Edman (N-terminal sequence) - if N-term. not blocked C-terminal sequence - not commonly performed Mass spectrometry-measured information

7. 150- 75- 40- 25- 18- 10- 6.5 6.05.55.04.5 pI MW x 10 3 2-D Gel Electrophoresis Excise separated protein “spots” In-gel trypsin digest Recover tryptic peptides m/z 500 717 1089 1272 1401 1547 1700 1857 2384 2791 1500 2500 3000 Peptide mass fingerprint by MALDI-TOF or LC-ESI-MS. Additional sequence information can be obtained by MS/MS. Protein Identification by Mass Spectrometry Protein identification by searching proteomic or genomic databases

8. Mass Spectrometry: A method to “weigh” molecules A simple measurement of mass is used to confirm the identity of a molecule, but it can be used for much more…… Other information can be inferred from a weight measurement. Post-translational modifications Post-translational modifications Molecular interactions Molecular interactions Shape Shape Sequence Sequence Physical dimensions Physical dimensions etc... etc...

9. Pre-Separation Mass Spectrometer for Proteomics Liquid Chromatography Mass AnalyzerIon Detector Time-of-Flight (TOF) Quadrupole TOF (QTOF) Ion Trap (IT) Fourier Transform- Ion Cyclotron Resonance (FT-ICR) Ion Source

10. The Nobel Prize in Chemistry 2002 "for the development of methods for identification and structure analyses of biological macromolecules" "for their development of soft desorption ionisation methods for mass spectrometric analyses of biological macromolecules" John Fenn Koichi Tanaka

11. Electrospray: Generation of aerosols and droplets

12. Electrospray Ionization (ESI) Multiple charging Multiple charging More charges for larger molecules More charges for larger molecules MW range > 150 kDa MW range > 150 kDa Liquid introduction of analyte Liquid introduction of analyte Interface with liquid separation methods, e.g. liquid chromatography Interface with liquid separation methods, e.g. liquid chromatography Tandem mass spectrometry (MS/MS) for protein sequencing Tandem mass spectrometry (MS/MS) for protein sequencing 5007009001100 mass/charge (m/z) 20+ 19+ 18+ 17+ 16+ 15+ 14+ 21+ 22+ highly charge droplets MS ESI

13. m/z 3323 3102 3543 (M+13H) 13+ ESI-MS of Large Proteins ESI-MS (Q-TOF) pH 7.5 46512 46048 mass (M+15H) 15+ (M+14H) 14+ (M+16H) 16+ distribution of multiply charged molecules

14. History of Electrospray Ionization Malcolm Dole demonstrated the production of intact oligomers of polystyrene up to MW 500,000 mass analysis of large ions was problematic John Fenn (Yale University) Chemical engineer - expert in supersonic molecular beams Began work on electrospray in 1981 Adapted ESI to operate on a more “conventional” mass spectrometer Recognized that multiply charged ions were produced by ESI Reduced the m/z range required

15. Electrospray process Analyte dissolved in a suitable solvent flows through a small diameter capillary tube Liquid in the presence of a high electric field generates a fine “mist” or aerosol spray of highly charged droplets 10 6 charges for 30 micron droplet

16. high voltage sample v1v1m1v1v1m1 v2v2m2v2v2m2 v3v3m3v3v3m3 m 1 m 2 m 3 detector drift region laser Matrix-assisted Laser Desorption/Ionization (MALDI) Time-of-Flight (TOF) Analyzer MALDI

17. 30811506087040590202110000129797 m/z 100 % Intensity 97430 98563 48658 36446 58309 MALDI Mass Spectrometry of Large Proteins MALDI-MS of rat MVP (M+H) + (M+2H) 2+

18. sample and matrix pulsed laser light 20 kV (sample stage or target) peptide/protein ions desorbed from matrix MALDI Developed by Tanaka (Japan) and Hillenkamp/Karas (Germany) Peptide/protein analyte of interest is co-crystallized on the MALDI target plate with an appropriate matrix small, highly conjugated organic molecules which strongly absorb energy at a particular wavelength Energy is transferred to analyte indirectly, inducing desorption from target surface Analyte is ionized by gas-phase proton transfer (perhaps from ionized matrix molecules)

19. MALDI matrices 4-hydroxy-  - cyanocinnamic acid (“alpha- cyano” or 4- HCCA)peptides 3,5-dimethoxy-4- hydroxycinnamic acid (sinapinic acid)proteins 2,5-dihydroxybenzoic acid (DHB) peptidesproteins peptides and proteins matrices for 337 nm irradiation

20. MALDI 337 nm irradiation is provided by a nitrogen (N 2 ) laser The target plate is inserted into the high vacuum region of the source and the sample is irradiated with a laser pulse. The matrix absorbs the laser energy and transfers energy to the analyte molecule. The molecules are desorbed and ionized during this stage of the process. MALDI is most commonly interfaced to a time-of-flight (TOF) mass spectrometer.

21. R. Aebersold and M. Mann, Nature (2003), 422, 198-207.

22. Time-of-Flight Mass Spectrometer v1v1m1v1v1m1 v2v2m2v2v2m2 v3v3m3v3v3m3 high voltage detector drift region (L) Principal of Operation of Linear TOF A time-of-flight mass spectrometer measures the mass-dependent time it takes ions of different masses to move from the ion source to the detector. This requires that the starting time (the time at which the ions leave the ion source) is well-defined. Recall that the kinetic energy of an ion is: where “ ” is ion velocity, “m” is mass, “e” is charge on electron, and “V” is electric field. The ion velocity,, is also the length of the flight path, L, divided by the flight time, t: Substituting this expression for into the kinetic energy relation, we can derive the working equation for the time-of-flight mass spectrometer: mass is proportional to (time) 2

23. MIRERICACVLALGMLTGFTHAFGSKDAAADGKPLVVTTIGMIADAVKNIAQGDVHLKGLMGP GVDPHLYTATAGDVEWLGNADLILYNGLHLETKMGEVFSKLRGSRLVVAVSETIPVSQRLSLE EAEFDPHVWFDVKLWSYSVKAVYESLCKLLPGKTREFTQRYQAYQQQLDKLDAYVRRKAQS LPAERRVLVTAHDAFGYFSRAYGFEVKGLQGVSTASEASAHDMQELAAFIAQRKLPAIFIESSI PHKNVEALRDAVQARGHVVQIGGELFSDAMGDAGTSEGTYVGMVTHNIDTIVAALAR Approaches for Protein Sequencing and Identification Enzymatic or chemical degradation MS/MS MS/MS “Top Down” “Bottom Up”

24. Identification of proteins from gels Proteins are separated first by high resolution two-dimensional polyacrylamide gel electrophoresis and then stained. At this point, to identify an individual or set of protein spots, several options can be considered by the researcher, depending on availability of techniques. For protein spots that appear to be relatively abundant (e.g., more than 1 pmol), traditional protein characterization methods may be employed For protein spots that appear to be relatively abundant (e.g., more than 1 pmol), traditional protein characterization methods may be employed. Methods such as amino acid analysis and Edman sequencing can be used to provide necessary protein identification information. With 2-DE, approximate molecular weight and isoelectric point characteristics are provided. Augmented with information on amino acid composition and/or amino-terminal sequence, a confident identification can be obtained. The sensitivity gains of using MS allows for the identification of proteins below the one pmol level and in many cases in the femtomole regime.

25. 150- 75- 40- 25- 18- 10- 6.5 6.05.55.04.5 pI MW x 10 3 2-D Gel Electrophoresis Excise separated protein “spots” In-gel trypsin digest Recover tryptic peptides m/z 500 717 1089 1272 1401 1547 1700 1857 2384 2791 1500 2500 3000 Peptide mass fingerprint by MALDI-TOF or LC-ESI-MS. Additional sequence information can be obtained by MS/MS. Protein Identification by Mass Spectrometry Protein identification by searching proteomic or genomic databases

26. Protein Cleavage For the application of mass spectrometry for protein identification, the protein bands/spots from a 2-D gel are excised and are exposed to a highly specific enzymatic cleavage reagent (e.g., trypsin cleaves on the C-terminal side of arginine and lysine residues). The resulting tryptic fragments are extracted from the gel slice and are then subjected to MS-methods. One of the major barriers to high throughput in the proteomic approach to protein identification is the “in-gel” proteolytic digestion and subsequent extraction of the proteolytic peptides from the gel. Common protocols for this process are often long and labor intensive. protein digestion robot

27. Protein cleavage - proteolysis and chemical methods

28. Mass spectrometry-based protein identification A mass spectrum of the resulting digest products produces a “peptide map” or a “peptide fingerprint”. The measured masses can be compared to theoretical peptide maps derived from database sequences for identification. There are a few choices of mass analysis that can be selected from this point, depending on available instrumentation and other factors. The resulting peptide fragments can be subjected to MALDI-MS or ESI-MS analysis. The measured molecular weights of the peptide fragments along with the specificity of the enzyme employed can be searched and compared against protein sequence databases A small aliquot of the digest solution can be directly analyzed by MALDI-MS to obtain a peptide map. The resulting sequence coverage (relative to the entire protein sequence) displayed from the total number of tryptic peptides observed in the MALDI mass spectrum can be quite high, i.e., greater than 80% of the sequence, although it can vary considerably depending on the protein, sample amount, etc. The measured molecular weights of the peptide fragments along with the specificity of the enzyme employed can be searched and compared against protein sequence databases using a number of computer searching routines available on the Internet.

29. Mass spectrum Theoretical mass spectrum Tryptic peptides Theoretical tryptic peptides Protein sequence SEMHIKHYTTKILGFRE EGDSCPLKQWDDSKIL VAVADKLLEYEEKILLF NSAKYLLDESSTYKLM HDDSV SEMHIKHYTTK ILGFR EEGDSCPLK QWDDSK ILVAVADK LLEYEEK ILLFNSAK YLLDESSTYK LMHDDSV Protein identification from peptide fragments

30. MALDI-MS of tryptic peptides 1000 1500200025003000 m/z 1116.67 1247.70 1287.73 1375.76 1424.85 1505.77 1665.89 1811.85 2005.07 2476.212550.52 2719.48 * * 1849.12 1574.20 KTVVIDFDI GLR RVVYDFV NVIQGDATLN QALIKDKRTE NLYILPASQT RDKDALTR DSDRILGILA SKRAENGE EPIKEHLLLT RYNPGRVSRG DMLSMEDVLE ILRLVGVI PEDQSVLRAS NQGEPVILDI NADAGKLLGER PFR ARIIVVTSGK GGVGKTTSSA AIATGLAQKG KKTVVIDFDI GLRNLDLIMG CERRVVYDFV NVIQGDATLN QALIKDKRTE NLYILPASQT RDKDALTREG VAKVLDDLKA MDFEFIVCDS PAGIETGALM ALYFADEAII TTNPEVSSVR DSDRILGILA SKSRRAENGE EPIKEHLLLT RYNPGRVSRG DMLSMEDVLE ILRIKLVGVI PEDQSVLRAS NQGEPVILDI NADAGKAYAD TVERLLGER PFRFIEEEKK GFLKRLFGG *trypsin autolysis all peaks are (M+H) +

31. ESI-MS and LC-MS for protein identification An approach for peptide mapping similar to MALDI-MS uses ESI-MS. A peptide map can be obtained by analysis of the peptide mixture by ESI-MS. An advantage of ESI is its ease of coupling to separation methodologies such as HPLC. Thus, alternatively, to reduce the complexity of the mixture, the peptides can be separated by HPLC with subsequent mass measurement by on-line ESI- MS. The measured masses can be compared to sequence databases. 400800120016002000 m/z 56789101112 Time (min) 9.4 9.8 8.4 8.9 7.7 6.8 6.2 0 100 Rel. Abund. 0 100 Rel. Abund. 965.3 629.0 LC-MS with ESI (M+2H) 2+ MW ~ 1928.6 Da

32. LC-MS/MS for protein identification An improvement in throughput of the overall method can be obtained by performing LC-MS/MS in the data dependant mode. As full scan mass spectra are acquired continuously in LC-MS mode, any ion detected with a signal intensity above a pre-defined threshold will trigger the mass spectrometer to switch over to MS/MS mode. Thus, the mass spectrometer switches back and forth between MS- (molecular mass information) and MS/MS mode (sequence information) in a single LC run. The data dependant scanning capability can dramatically increase the capacity and throughput for protein identification. 400800120016002000 m/z 56789101112 Time (min) 9.4 9.8 8.4 6.8 965.3 629.0 LC-MS (M+2H) 2+ 40060080010001200140016001800 m/z 1261.4 1374.5 668.4 838.5 1474.4 y4y4 b3b3 b5b5 b6b6 b8b8 y8y8 y9y9 y 10 y 11 y 12 y 13 y 14 LC-MS/MS MS/MS

33. A B C D E A B C D E A B C D A B C A B A m/z Peptide sequencing by mass spectrometry Peptide molecules are fragmented by collisionally activated dissociation (CAD) collisions with neutral background gas molecules (nitrogen, argon, etc) typically dissociate by cleavage of -CO-NH- bond N-term. C-term. N-terminal product ions

34. A B C D E D B C E C D E D E E m/z C-terminal product ions Ideally, one can measure the spacings between product ion peaks to deduce the sequence if each amide bond dissociates with equal probability if only a single amide bond fragments for each molecule if only C-terminal or N-terminal products ions are formed In reality, this is not the case… In reality, this is not the case… Peptide sequencing by mass spectrometry

35. H 2 N - C - C - N - C - C - N - C - C - N - C - COOH OOO HHHHHHH R1R1 R2R2 R3R3 R4R4 N-terminal fragments C-terminal fragments b1b1 y3y3 b2b2 y2y2 b3b3 y1y1 Nomenclature for MS Sequencing of Peptides Klaus Biemann, MIT subscript denotes the number of residues contained in product ion

36. Nomenclature for MS Sequencing of Peptides Low-energy collisions promote fragmentation of a peptide primarily along the peptide backbone Peptide fragmentation which maintains the charge on the C terminus is designated a y-ion Fragmentation which maintains the charge on the N terminus is designated a b-ion Low energy collisions: ion trap, QQQ, QTOF, FT-ICR High energy collisions: TOF-TOF cleavage of amino acid side chain bonds (d-ion and w-ion) differentiate Leu vs. Ile

37. 40060080010001200140016001800 m/z 0 100 Rel. Abund. 1261.4 1091.5 1374.5 668.4 838.5 555.4 990.5 1474.4 y4y4 b3b3 b4b4 b5b5 b6b6 b7b7 b8b8 b 13 b 12 b 11 b 10 b9b9 b 14 b 15 b 16 b 17 y5y5 y6y6 y7y7 y8y8 y9y9 y 10 y 11 y 12 y 13 y 14 LVDKVIGITNEEAISTAR b 3-17 y 4-14 242 259 Cysteine Synthase A MS/MS of 2+ charged tryptic peptides yield (often) 1+ charged product ions (but 2+ charged products can be observed as well) Peptide Sequencing by Mass Spectrometry mixture of b-ions and y- ions are present

38. Computer-based Sequence Searching Strategies A list of experimentally determined masses is compared to lists of computer-generated theoretical masses prepared from a database of protein primary sequences. With the current exponential growth in the generation of genomic data, these databases are expanding every day. There are typically three types of search strategies employed: searching with peptide fingerprint data searching with sequence data searching with raw MS/MS data. One limiting factor that must be considered for all of the approaches is that they can only identify proteins that have been identified and reside within an available database, or very homologous to one that resides in the database.

39. Searching with Peptide Fingerprints The majority of the available search engines allow one to define certain experimental parameters to optimize a particular search. Minimum number of peptides to be matched Allowable mass error Monoisotopic versus average mass data Mass range of starting protein Type of protease used for digestion Information about potential protein modification, such as N- and C-terminal modification, carboxymethylation, oxidized methionines, etc.

40. Searching with Peptide Fingerprints Most protein databases contain primary sequence information only Any shift in mass incorporated into the primary sequence as a result of post-translational modification will result in an experimental mass that is in disagreement with the theoretical mass. Modifications such as glycation and phosphorylation can result in missed identifications. A single amino acid substitution can shift the mass of a peptide to such a degree that even a protein with a great deal of homology with another in the database can not be identified.

41. Searching with Peptide Fingerprints A number of factors affect the utility of peptide fingerprinting. experimental mass accuracy The greater the experimental mass accuracy, the narrower you can set your search tolerances, thereby increasing your confidence in the match, and decreasing the number of "false positive" responses. A common practice used to increase mass accuracy in peptide fingerprinting is to employ an autolysis fragment from the proteolytic enzyme as an internal standard to calibrate a MALDI mass spectrum. Peptide fingerprinting is also amenable to the identification of proteins in complex mixtures. Peptides generated from the digest of a protein mixture will simply return two or more results that are a "good" fit. Peptides that are "left over" in a peptide fingerprint after the identification of one component can be resubmitted for the possible identification of another component.

42. Web addresses of some representative internet resources for protein identification from mass spectrometry data

43. Mascot Among the first programs for identifying proteins by peptide mass fingerprinting, MOWSE, developed out of a collaboration between Imperial Cancer Research Fund (ICRF) and SERC Daresbury Laboratory, UK. The name chosen was an acronym of Molecular Weight Search. The MOWSE databases were fully indexed so as to allow very rapid searching and retrieval of sequence data. Subsequently, the software was further developed and renamed Mascot. Licensed and distributed by Matrix Science Ltd. Specialized tools include Peptide Mass Fingerprint, Sequence Query, and MS/MS Ion Search. Search output Web-based. Good visual representation of search quality (graphical probability chart). Simple graphical user interface. Reports MOWSE scores as a quantitative measure of search quality.

44. Mowse Scoring Mowse uses empirically determined factors to assign a statistical weight to each individual peptide match Rather than just counting the number of matching peptides, Mowse uses empirically determined factors to assign a statistical weight to each individual peptide match. Rapid identification of proteins by peptide-mass fingerprinting. Curr. Biol. 3:327, 1993.) Scoring scheme assigns more weight to matches of higher molecular weight peptides (more discriminating). Compensates for the non-random distribution of fragment molecular weights in proteins of different sizes. Was first protein identification program to recognize that the relative abundance of peptides of a given length in a proteolytic digest depends on the lengths of both peptide and protein. Developed for MALDI peptide mass fingerprinting. Probability-Based Mowse Mascot incorporates a probability-based enhanced Mowse algorithm, described in Perkins et al. (Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20:3551-3567, 1999). A simple rule can be used to judge whether a result is significant or not. Different types of matching (peptide masses and fragment ions) can be combined in a single search.

45. Databases Three components are required for database searching support of proteomics: MALDI or MS/MS data, the algorithms used to search protein databases with the MALDI or MS/MS data, and the protein databases themselves. The protein databases can be as small as one protein, can be large, public domain databases of all known and predicted proteins, or may be predicted open reading frames based on genomic sequence. A major challenge for database searching is that these protein databases are constantly changing, making database search results potentially obsolete as new entries are added that better fit the MALDI or MS data. A major challenge for database searching is that these protein databases are constantly changing, making database search results potentially obsolete as new entries are added that better fit the MALDI or MS data. Even as genomes are completed there is still flux as new coding regions are identified and novel mechanisms of increased translational complexity are better understood, such as alternative splice products, RNA editing, and ribosome slippage leading to novel, unexpected translation products.

46. Databases NCBI non-redundant (NCBInr) Non-redundant database from the National Center for Biotechnology Information for use with their search tools BLAST and Entrez; comprised of translated sequences from the Genbank /EMBL/DDBJ consortium, SwissProt, Protein Information Resource (PIR), and Brookhaven Protein Data Bank (PDB). New releases are published bimonthly while updates occur daily. OWL OWL is comprised of Swiss-Prot, PIR, translated Genbank, and NRL-3D (PDB). All sequences are compared to Swiss-Prot to remove identical and “trivially different “ sequences. Has not been updated since May, 1999. SWISSPROT While SwissProt contains only a subset of proteins, the proteins in this database are much better annotated and the sequences are much more reliable than those available in any other database. MSDB Comprehensive, non-identical protein sequence database maintained by the Proteomics Department at the Hammersmith Campus of Imperial College London. Designed specifically for MS applications.

47. Databases EST Clusters (dBEST) Division of GenBank that contains "single-pass" cDNA sequences, or Expressed Sequence Tags (EST’s), from a number of organisms. EST’s are relatively short, usually 3’ end sequences from isolated mRNA. EST’s tend to be highly redundant and the sequence is much lower quality than from other sources. An advantage to using these EST’s is that they represent only expressed sequences (no introns) and include alternative splice variants; their length, redundancy, and low quality are far improved by using clustered EST’s, such as the Compugen clusters. The EST database has some redundancy because it contains all possible combinations of alternative splice products, and so it can be very large (and slow to search). During a Mascot search, the nucleic acid sequences are translated in all six reading frames. dbEST is a very large database, and is divided into three sections: EST_human, EST_mouse, and EST_others. Even so, searches of these databases take far longer than a search of one of the non-redundant protein databases. You should only search an EST database if a search of a protein database has failed to find a match.

48. MALDI-MS peptide fingerprint (tryptic digest of a single protein) 1000 1500200025003000 m/z 1116.67 1247.70 1287.73 1375.76 1424.85 1505.77 1665.89 1811.85 2005.07 2476.212550.52 2719.48 * * 1849.12 1574.20 *trypsin autolysis all peaks are (M+H) +

49. Mascot (Matrix Science) for peptide mass fingerprints enter peak list

50. Mascot (Matrix Science) for peptide mass fingerprints possible identification

51. Mascot (Matrix Science) for peptide mass fingerprints list of all possible matches get more info on probable proteins

52. Mascot (Matrix Science) for peptide mass fingerprints

53. tryptic peptides that matched peptides that did not match

54. Mascot (Matrix Science) for peptide mass fingerprints tryptic peptides in protein sequence better mass accuracy improves identification process

55. LC-MS/MS for protein identification To provide further confirmation of the identification, if a tandem mass spectrometer (MS/MS) is available, peptide ions can be dissociated in the mass spectrometer to provide direct sequence information. Product ions from an MS/MS spectrum can be compared to available sequences using powerful software toolsl. LC-MS/MS analysis included two discrete steps For a single sample, LC-MS/MS analysis included two discrete steps: (a) LC-MS peptide mapping to identify peptide ions from the digestion mixture and to deduce their molecular weights, and (b) LC-MS/MS of the previously detected peptides to obtain sequence information for protein identification.

56. Automated LC-MS/MS and database searching Current mass spectral technology permits the generation of MS/MS data at an unprecedented rate. Prior to the generation of powerful computer-based database searching strategies, the largest bottleneck in protein identification was the manual interpretation of this MS/MS data to extract the sequence information. Today, many computer-based search strategies that employ MS/MS data require no operator interpretation at all. Analogous to the approach described for peptide fingerprinting, these programs take the individual protein entries in a database and electronically "digest" them to generate a list of theoretical peptides for each protein. However, in the use of MS/MS data, these theoretical peptides are further manipulated to generate a second level of lists which contain theoretical fragment ion masses that would be generated in the MS/MS experiment for each theoretical peptide.

57. Automated LC-MS/MS and database searching These programs simply compare the list of experimentally determined fragment ion masses from the MS/MS experiment of the peptide of interest with the theoretical fragment ion masses generated by the computer program. The recent advent of data-dependant scanning functions has permitted the unattended acquisition of MS/MS data. An example of a raw MS/MS data searching program that takes particular advantage of this ability is SEQUEST. SEQUEST will input the data from a data-dependant LC/MS chromatogram and automatically strip out all of the MS/MS information for each individual peak, and submit it for database searching using the strategy discussed above. Each peak is treated as a separate data file, making it especially useful for the on-line separation and identification of individual components in a protein mixture. SEQUEST cross-correlates uninterpreted MS/MS mass spectra of peptides from protein/nucleotide databases. The software can analyze a single spectrum or an entire LC-MS/MS peptide map. No user interpretation of MS/MS spectra is involved.

58. 9001000 m/z 50 100 475.3 588.3 456.7 325.2 701.3815.4 410.3 212.1 851.5 912.5 Proteolytic Digest Time (min) 2530354045505560 0 50 10041.63 54.28 60.16 62.59 49.20 38.27 46.75 33.59 29.02 MS/MS Match? Experimental Fragment Masses HPLC-MS Theoretical Peptide LPNLIYHR Theoretical Fragment Masses

59. Link et al. Nature Biotechnology 17, 676 (1999) Direct identification of proteins using mass spectrometry Removes the requirement to separate proteins by electrophoresis, etc MudPIT: multidimensional protein identification technology, or “Shotgun” approach Protein lysate is digested with trypsin The peptide mixture is loaded onto a strong cation exchange (SCX) column (to separate on the basis of charge). A discrete fraction of peptides is displaced from the SCX column using a salt step gradient to a reversed-phase (RP) column (to separate on the basis of hydrophobicity). This fraction is eluted from the RP column into the MS. This iterative process is repeated, obtaining the fragmentation patterns of peptides in the original peptide mixture. MS/MS spectra are used to identify the proteins in the original protein complex.

60. Large-scale analysis of the yeast proteome by MudPIT Yates and coworkers, Nature Biotech. (2001) 19, 242-247 Assigned 5,540 peptides to MS spectra leading to the identification of 1,484 proteins from the S. cerevisiae proteome Of 6,216 ORFs in yeast genome, 83% have CAI values between 0 and 0.20 (i.e., predicted to be present at low levels) (Fig. A) MudPIT data: 791 or 53.3% of the proteins identified have a CAI of <0.2 (1.7 peptides per protein) (Fig. B) Number of peptides per protein increases with increasing CAI (Fig. C)

61. MIRERICACVLALGMLTGFTHAFGSKDAAADGKPLVVTTIGMIADAVKNIAQGDVHLKGLMGP GVDPHLYTATAGDVEWLGNADLILYNGLHLETKMGEVFSKLRGSRLVVAVSETIPVSQRLSLE EAEFDPHVWFDVKLWSYSVKAVYESLCKLLPGKTREFTQRYQAYQQQLDKLDAYVRRKAQS LPAERRVLVTAHDAFGYFSRAYGFEVKGLQGVSTASEASAHDMQELAAFIAQRKLPAIFIESSI PHKNVEALRDAVQARGHVVQIGGELFSDAMGDAGTSEGTYVGMVTHNIDTIVAALAR Approaches for Protein Sequencing and Identification MS/MS “Top Down” The molecular mass of an intact protein defines the native covalent state of a gene’s product, including the effects of post-transcriptional/translational modifications, and associated heterogeneity, that are modulated by the actions of other gene products. Moreover, the fragmentation pattern from large proteins can generate sufficient information for identification from sequence databases, particularly when combined with accurate mass measurements of both the intact molecule and its product ions.

62. PE E E P T T I D F R A G M N In-Source Decay (ISD) for Protein Sequencing PE P P E P T P E P T I P E P T I D P E E P T I D cut...TIDE... Peptides and large proteins can be fragmented by ISD Fragmentation occurs in the MALDI ion source not generally well controlled Reflectron TOF not necessary (linear TOF sufficient to measure product ions Complete sequence information not present, but extensive stretches of sequence from the N- and/or C-termini observed

63. 5000 5000 10000 10000 15000 15000 20000 20000 25000 25000 m/z Protein 1 Protein 2 Molecular Weight Information Sequence Information (In-Source Decay) MALDI-ISD-TOF Mass Spectrometry of Proteins

64. 2500 2500 3000 3000 3500 3500 4000 4000 m/z AL Y GG E GF E D H R L K/Q E D T PW? WP? AL F G D E GF E D H R V A F D L/N Sequence information from the N-terminus MALDI-ISD-TOF Mass Spectrometry of Proteins ISD generally yields c-ions or z-ions

65. (top) ESI-QqTOF-MS of transferrin and (bottom) ESI- MS/MS of 36+ charged molecule. Sequence specific b n -products from residues 56- 69 are generated. In addition, a series of larger products appears at higher m/z above the precursor molecule position that range in size from 69-73 kDa that are consistent for the y n - complement to the b n - products observed in the lower m/z region of the spectrum. (Thevis et al, J. Am. Soc. Mass Spectrom. 2003, 14, 635). Top-down sequencing of transferrin by ESI-MS/MS

66. Masses and compositions of commonly occuring amino acid residues