1. Mass Spectrometry: Methods & Theory

2. Proteomics Tools Molecular Biology Tools Separation & Display Tools Protein Identification Tools Protein Structure Tools

3. Mass Spectrometry Needs Ionization-how the protein is injected in to the MS machine Separation-Mass and Charge is determined Activation-protein are broken into smaller fragments (peptides/AAs) Mass Determination-m/z ratios are determined for the ionized protein fragments/peptides

4. Protein Identification 2D-GE + MALDI-MS –Peptide Mass Fingerprinting (PMF) 2D-GE + MS-MS –MS Peptide Sequencing/Fragment Ion Searching Multidimensional LC + MS-MS –ICAT Methods (isotope labelling) –MudPIT (Multidimensional Protein Ident. Tech.) 1D-GE + LC + MS-MS De Novo Peptide Sequencing

5. Mass Spectrometry (MS) Introduce sample to the instrument Generate ions in the gas phase Separate ions on the basis of differences in m/z with a mass analyzer Detect ions

6. How does a mass spectrometer work? Ionization method –MALDI –Electrospray (Proteins must be charged and dry) Mass analyzer –MALDI-TOF MW –Triple Quadrapole AA seq –MALDI-QqTOF AA seq and MW –QqTOF AA seq and protein modif. Create ionsSeparate ions Detect ions Mass spectrum Database analysis

7. Generalized Protein Identification by MS Artificial spectra built Artificially trypsinated Database of sequences (i.e. SwissProt) Spot removed from gel Fragmented using trypsin Spectrum of fragments generated MATC H Library

8. Methods for protein identification

9. MS Principles Different elements can be uniquely identified by their mass

10. MS Principles Different compounds can be uniquely identified by their mass CH 3 CH 2 OH N OH HO -CH 2 - -CH 2 CH-NH 2 COOH HO Butorphanol L-dopa Ethanol MW = 327.1 MW = 197.2 MW = 46.1

11. Mass Spectrometry Analytical method to measure the molecular or atomic weight of samples

12. Weighing proteins Weighing proteins A mass spectrometer creates charged particles (ions) from molecules. Common way is to add or take away an ions: NaCl + e -  NaCl - NaCl  NaCl + + e - It then analyzes those ions to provide information about the molecular weight of the compound and its chemical structure.

13. Mass Spectrometry For small organic molecules the MW can be determined to within 5 ppm or 0.0005% which is sufficiently accurate to confirm the molecular formula from mass alone For large biomolecules the MW can be determined within an accuracy of 0.01% (i.e. within 5 Da for a 50 kD protein) Recall 1 dalton = 1 atomic mass unit (1 amu)

14. MS History JJ Thomson built MS prototype to measure m/z of electron, awarded Nobel Prize in 1906 MS concept first put into practice by Francis Aston, a physicist working in Cambridge England in 1919 Designed to measure mass of elements Aston Awarded Nobel Prize in 1922

15. MS History 1948-52 - Time of Flight (TOF) mass analyzers introduced 1955 - Quadrupole ion filters introduced by W. Paul, also invents the ion trap in 1983 (wins 1989 Nobel Prize) 1968 - Tandem mass spectrometer appears Mass spectrometers are now one of the MOST POWERFUL ANALYTIC TOOLS IN CHEMISTRY

16. MS Principles Find a way to “charge” an atom or molecule (ionization) Place charged atom or molecule in a magnetic field or subject it to an electric field and measure its speed or radius of curvature relative to its mass-to-charge ratio (mass analyzer) Detect ions using microchannel plate or photomultiplier tube

17. Mass Spec Principles Ionizer Sample + _ Mass Analyzer Detector

18. How does a mass spectrometer work? Ionization method –MALDI –Electrospray (Proteins must be charged and dry) Mass analyzer –MALDI-TOF MW –Triple Quadrapole AA seq –MALDI-QqTOF AA seq and MW –QqTOF AA seq and protein modif. Create ionsSeparate ions Detect ions Mass spectrum Database analysis

19. Mass spectrometers Time of flight (TOF) (MALDI)Time of flight (TOF) (MALDI) –Measures the time required for ions to fly down the length of a chamber. –Often combined with MALDI (MALDI-TOF) Detections from multiple laser bursts are averaged. Multiple laser Tandem MS- MS/MS -separation and identification of compounds in complex mixtures - induce fragmentation and mass analyze the fragment ions. - Uses two or more mass analyzers/filters separated by a collision cell filled with Argon or Xenon Different MS-MS configurations –Quadrupole-quadrupole (low energy) –Magnetic sector-quadrupole (high) –Quadrupole-time-of-flight (low energy) –Time-of-flight-time-of-flight (low energy)

20. Typical Mass Spectrometer

21. LC/LC-MS/MS-Tandem LC, Tandem MS

22. Typical Mass Spectrum Characterized by sharp, narrow peaks X-axis position indicates the m/z ratio of a given ion (for singly charged ions this corresponds to the mass of the ion) Height of peak indicates the relative abundance of a given ion (not reliable for quantitation) Peak intensity indicates the ion’s ability to desorb or “fly” (some fly better than others)

23. m/z ratio: Molecular weight divided by the charge on this protein All proteins are sorted based on a mass to charge ratio (m/z)

24. Typical Mass Spectrum aspirin Relative Abundance singly 120 m/z-for singly charged ion this is the mass

25. Resolution & Resolving Power Width of peak indicates the resolution of the MS instrument The better the resolution or resolving power, the better the instrument and the better the mass accuracy Resolving power is defined as: M is the mass number of the observed mass (  M) is the difference between two masses that can be separated MM M

26. Resolution in MS

27. QTOF 783.455 784.465 785.475 783.6

28. Mass Spectrometer Schematic Inlet Ion Source Mass Filter Detector Data System High Vacuum System Turbo pumps Diffusion pumps Rough pumps Rotary pumps Sample Plate Target HPLC GC Solids probe MALDI ESI IonSpray FAB LSIMS EI/CI TOF Quadrupole Ion Trap Mag. Sector FTMS Microch plate Electron Mult. Hybrid Detec. PC’s UNIX Mac

29. Different Ionization Methods Electron Impact (EI - Hard method) –small molecules, 1-1000 Daltons, structure Fast Atom Bombardment (FAB – Semi-hard) –peptides, sugars, up to 6000 Daltons Electrospray Ionization (ESI - Soft) –peptides, proteins, up to 200,000 Daltons Matrix Assisted Laser Desorption ( MALDI -Soft) –peptides, proteins, DNA, up to 500 kD

30. Electron Impact Ionization Sample introduced into instrument by heating it until it evaporates Gas phase sample is bombarded with electrons coming from rhenium or tungsten filament (energy = 70 eV) Molecule is “shattered” into fragments (70 eV >> 5 eV bonds) Fragments sent to mass analyzer

32. EI Fragmentation of CH 3 OH CH 3 OHCH 3 OH + CH 3 OHCH 2 O=H + + H CH 3 OH + CH 3 + OH CHO=H + + HCH 2 O=H + Why wouldn’t Electron Impact be suitable for analyzing proteins?

33. Why You Can’t Use EI For Analyzing Proteins EI shatters chemical bonds Any given protein contains 20 different amino acids EI would shatter the protein into not only into amino acids but also amino acid sub- fragments and even peptides of 2,3,4… amino acids Result is 10,000’s of different signals from a single protein -- too complex to analyze

34. Soft Ionization Methods 337 nm UV laser MALDI cyano-hydroxy cinnamic acid Gold tip needle Fluid (no salt) ESI + _

35. Soft Ionization Soft ionization techniques keep the molecule of interest fully intact Electro-spray ionization first conceived in 1960’s by Malcolm Dole but put into practice in 1980’s by John Fenn (Yale) MALDI first introduced in 1985 by Franz Hillenkamp and Michael Karas (Frankfurt) Made it possible to analyze large molecules via inexpensive mass analyzers such as quadrupole, ion trap and TOF

37. Ionization methods Electrospray mass spectrometry (ESI-MS) –Liquid containing analyte is forced through a steel capillary at high voltage to electrostatically disperse analyte. Charge imparted from rapidly evaporating liquid. Matrix-assisted laser desorption ionization (MALDI) –Analyte (protein) is mixed with large excess of matrix (small organic molecule) –Irradiated with short pulse of laser light. Wavelength of laser is the same as absorbance max of matrix.

38. Electrospray Ionization Sample dissolved in polar, volatile buffer (no salts) and pumped through a stainless steel capillary (70 - 150  m) at a rate of 10- 100  L/min Strong voltage (3-4 kV) applied at tip along with flow of nebulizing gas causes the sample to “nebulize” or aerosolize Aerosol is directed through regions of higher vacuum until droplets evaporate to near atomic size (still carrying charges)

39. Electrospray (Detail)

40. Electrospray Ionization Can be modified to “nanospray” system with flow < 1  L/min Very sensitive technique, requires less than a picomole of material Strongly affected by salts & detergents Positive ion mode measures (M + H) + (add formic acid to solvent) Negative ion mode measures (M - H) - (add ammonia to solvent)

41. Positive or Negative Ion Mode? If the sample has functional groups that readily accept H+ (such as amide and amino groups found in peptides and proteins) then positive ion detection is used-PROTEINS If a sample has functional groups that readily lose a proton (such as carboxylic acids and hydroxyls as found in nucleic acids and sugars) then negative ion detection is used-DNA

42. Matrix-Assisted Laser Desorption Ionization 337 nm UV laser MALDI cyano-hydroxy cinnamic acid

43. MALDI Sample is ionized by bombarding sample with laser light Sample is mixed with a UV absorbant matrix (sinapinic acid for proteins, 4- hydroxycinnaminic acid for peptides) Light wavelength matches that of absorbance maximum of matrix so that the matrix transfers some of its energy to the analyte (leads to ion sputtering)

44. HT Spotting on a MALDI Plate

45. MALDI Ionization + + + + - - - + + + + - - - -+ + Analyte Matrix Laser + + + Absorption of UV radiation by chromophoric matrix and ionization of matrix Dissociation of matrix, phase change to super- compressed gas, charge transfer to analyte molecule Expansion of matrix at supersonic velocity, analyte trapped in expanding matrix plume (explosion/”popping”) + + +

46. MALDI Unlike ESI, MALDI generates spectra that have just a singly charged ion Positive mode generates ions of M + H Negative mode generates ions of M - H Generally more robust that ESI (tolerates salts and nonvolatile components) Easier to use and maintain, capable of higher throughput Requires 10  L of 1 pmol/  L sample

47. Principal for MALDI-TOF MASS

49. MALDI = SELDI 337 nm UV laser MALDI cyano-hydroxy cinnaminic acid

50. MALDI/SELDI Spectra Normal Tumor

51. Mass Filter Mass Spectrometer Schematic Inlet Ion Source Detector Data System High Vacuum System Turbo pumps Diffusion pumps Rough pumps Rotary pumps Sample Plate Target HPLC GC Solids probe MALDI ESI IonSpray FAB LSIMS EI/CI TOF Quadrupole Ion Trap Mag. Sector FTMS Microch plate Electron Mult. Hybrid Detec. PC’s UNIX Mac

52. Different Mass Analyzers Magnetic Sector Analyzer (MSA) –High resolution, exact mass, original MA Quadrupole Analyzer (Q) –Low (1 amu) resolution, fast, cheap Time-of-Flight Analyzer (TOF) –No upper m/z limit, high throughput Ion Trap Mass Analyzer (QSTAR) –Good resolution, all-in-one mass analyzer Ion Cyclotron Resonance (FT-ICR) –Highest resolution, exact mass, costly

53. Different Types of MS ESI-QTOF –Electrospray ionization source + quadrupole mass filter + time-of-flight mass analyzer MALDI-QTOF –Matrix-assisted laser desorption ionization + quadrupole + time-of-flight mass analyzer Both separate by MW and AA seq

54. Different Types of MS GC-MS - Gas Chromatography MS –separates volatile compounds in gas column and ID’s by mass LC-MS - Liquid Chromatography MS –separates delicate compounds in HPLC column and ID’s by mass MS-MS - Tandem Mass Spectrometry –separates compound fragments by magnetic field and ID’s by mass LC/LC-MS/MS-Tandem LC and Tandem MS –Separates by HPLC, ID’s by mass and AA sequence

55. Magnetic Sector Analyzer

56. Quadrupole Mass Analyzer A quadrupole mass filter consists of four parallel metal rods with different charges Two opposite rods have an applied + potential and the other two rods have a - potential The applied voltages affect the trajectory of ions traveling down the flight path For given dc and ac voltages, only ions of a certain mass-to-charge ratio pass through the quadrupole filter and all other ions are thrown out of their original path

57. Quadrupole Mass Analyzer

58. Q-TOF Mass Analyzer TOF NANOSPRAY TIP ION SOURCE HEXAPOLE COLLISION CELL HEXAPOLE QUADRUPOLE MCP DETECTOR REFLECTRON SKIMMER PUSHER

59. Mass Spec Equation (TOF) m z 2Vt 2 = m = mass of ionL = drift tube length z = charge of iont = time of travel V = voltage L2L2

60. Ion Trap Mass Analyzer Ion traps are ion trapping devices that make use of a three- dimensional quadrupole field to trap and mass- analyze ions invented by Wolfgang Paul (Nobel Prize1989) Offer good mass resolving power

61. FT-ICR Fourier-transform ion cyclotron resonance Uses powerful magnet (5-10 Tesla) to create a miniature cyclotron Originally developed in Canada (UBC) by A.G. Marshal in 1974 FT approach allows many ion masses to be determined simultaneously (efficient) Has higher mass resolution than any other MS analyzer available

62. FT-Ion Cyclotron Analzyer

63. Current Mass Spec Technologies Proteome profiling/separation –2D SDS PAGE - identify proteins –2-D LC/LC - high throughput analysis of lysates (LC = Liquid Chromatography) –2-D LC/MS (MS= Mass spectrometry) Protein identification –Peptide mass fingerprint –Tandem Mass Spectrometry (MS/MS) Quantative proteomics –ICAT (isotope-coded affinity tag) –ITRAQ

64. 2D - LC/LC Study protein complexes without gel electrophoresis Peptides all bind to cation exchange column Peptides are separated by hydrophobicity on reverse phase column Successive elution with increasing salt gradients separates peptides by charge Complex mixture is simplified prior to MS/MS by 2D LC (trypsin)

65. 2D - LC/MS

66. Peptide Mass Fingerprinting (PMF)

67. Peptide Mass Fingerprinting Used to identify protein spots on gels or protein peaks from an HPLC run Depends of the fact that if a peptide is cut up or fragmented in a known way, the resulting fragments (and resulting masses) are unique enough to identify the protein Requires a database of known sequences Uses software to compare observed masses with masses calculated from database

68. Principles of Fingerprinting >Protein 1 acedfhsakdfqea sdfpkivtmeeewe ndadnfekqwfe >Protein 2 acekdfhsadfqea sdfpkivtmeeewe nkdadnfeqwfe >Protein 3 acedfhsadfqeka sdfpkivtmeeewe ndakdnfeqwfe Sequence Mass ( M+H ) Tryptic Fragments 4842.054842.054842.05 acedfhsak dfgeasdfpk ivtmeeewendadnfek gwfe acek dfhsadfgeasdfpk ivtmeeewenk dadnfeqwfe acedfhsadfgek asdfpk ivtmeeewendak dnfegwfe

69. Principles of Fingerprinting >Protein 1 acedfhsakdfqea sdfpkivtmeeewe ndadnfekqwfe >Protein 2 acekdfhsadfqea sdfpkivtmeeewe nkdadnfeqwfe >Protein 3 acedfhsadfqeka sdfpkivtmeeewe ndakdnfeqwfe Sequence Mass ( M+H ) Mass Spectrum 4842.05

70. Predicting Peptide Cleavages http://ca.expasy.org/tools/peptidecutter/

71. http://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#Tryps

72. Rules Protease Cleavage Rules TrypsinXXX[KR]--[!P]XXX ChymotrypsinXX[FYW]--[!P]XXX Lys CXXXXXK-- XXXXX Asp N endoXXXXXD-- XXXXX CNBrXXXXXM--XXXXX K-Lysine, R-Arginine, F-Phenylalanine, Y-Tyrosine, W-Tryptophan,D-Aspartic Acid, M-Methionine, P-Proline Sometimes inhibition occurs

73. Why Trypsin? Robust, stable enzyme Works over a range of pH values & Temp. Quite specific and consistent in cleavage Cuts frequently to produce “ideal” MW peptides Inexpensive, easily available/purified Does produce “autolysis” peaks (which can be used in MS calibrations) –1045.56, 1106.03, 1126.03, 1940.94, 2211.10, 2225.12, 2283.18, 2299.18

74. Digest with specific protease >RBME00320 Contig0311_1089618_1091255 EC-mopA 60 KDa chaperonin GroEL MAAKDVKFGR TAREKMLRGV DILADAVKVT LGPKGRNVVI EKSFGAPRIT KDGVSVAKEV ELEDKFENMG AQMLREVASK TNDTAGDGTT TATVLGQAIV QEGAKAVAAG MNPMDLKRGI DLAVNEVVAE LLKKAKKINT SEEVAQVGTI SANGEAEIGK MIAEAMQKVG NEGVITVEEA KTAETELEVV EGMQFDRGYL SPYFVTNPEK MVADLEDAYI LLHEKKLSNL QALLPVLEAV VQTSKPLLII AEDVEGEALA TLVVNKLRGG LKIAAVKAPG FGDCRKAMLE DIAILTGGQV ISEDLGIKLE SVTLDMLGRA KKVSISKENT TIVDGAGQKA EIDARVGQIK QQIEETTSDY DREKLQERLA KLAGGVAVIR VGGATEVEVK EKKDRVDDAL NATRAAVEEG IVAGGGTALL RASTKITAKG VNADQEAGIN IVRRAIQAPA RQITTNAGEE ASVIVGKILE NTSETFGYNT ANGEYGDLIS LGIVDPVKVV RTALQNAASV AGLLITTEAM IAELPKKDAA PAGMPGGMGG MGGMDF 546 aa60 kDa; 57 461 Da pI = 4.75

75. Digest with specific protease Trypsin yields 47 peptides (theoretically) 501.3533.3544.3545.3614.4634.3 674.3675.4701.4726.4822.4855.5 861.4879.4921.5953.4974.5988.5 1000.61196.61217.61228.51232.61233.7 1249.61249.61344.71455.81484.61514.8 1582.91583.91616.81726.71759.91775.9 1790.61853.91869.92286.22302.22317.2 2419.22526.42542.43329.64211.4 Peptide masses in Da: http://us.expasy.org/tools/peptide-mass.html

76. Digest with trypsin In practice.......see far fewer by mass spec - possibly incomplete digest (we allow 1 miss) - lose peptides during each manipulation washes during digestion washes during cleanup step some peptides will not ionize well some signals (peaks) are poor low intensity; lack resolution

77. What Are Missed Cleavages? >Protein 1 acedfhsakdfqea sdfpkivtmeeewe ndadnfekqwfe SequenceTryptic Fragments ( no missed cleavage ) acedfhsak (1007.4251) dfgeasdfpk (1183.5266) ivtmeeewendadnfek (2098.8909) gwfe (609.2667) Tryptic Fragments ( 1 missed cleavage ) acedfhsak (1007.4251) dfgeasdfpk (1183.5266) ivtmeeewendadnfek 2098.8909) gwfe (609.2667) acedfhsakdfgeasdfpk (2171.9338) ivtmeeewendadnfekgwfe (2689.1398) dfgeasdfpkivtmeeewendadnfek (3263.2997)

78. Calculating Peptide Masses Sum the monoisotopic residue masses Monoisotopic Mass: the sum of the exact or accurate masses of the lightest stable isotope of the atoms in a molecule Add mass of H 2 O (18.01056) Add mass of H + (1.00785 to get M+H) If Met is oxidized add 15.99491 If Cys has acrylamide adduct add 71.0371 If Cys is iodoacetylated add 58.0071 Other modifications are listed at –http://prowl.rockefeller.edu/aainfo/deltamassv2.htmlhttp://prowl.rockefeller.edu/aainfo/deltamassv2.html 1 H-1.007828503 amu 12 C-12 2 H-2.014017780 amu 13 C-13.00335, 14 C-14.00324

79. Masses in MS Monoisotopic mass is the mass determined using the masses of the most abundant isotopes Average mass is the abundance weighted mass of all isotopic components

80. Mass Calculation (Glycine) NH 2 —CH 2 —COOH R 1 —NH—CH 2 —CO—R 3 Amino acid Residue Monoisotopic Mass 1 H = 1.007825 12 C = 12.00000 14 N = 14.00307 16 O = 15.99491 Glycine Amino Acid Mass 5xH + 2xC + 2xO + 1xN = 75.032015 amu Glycine Residue Mass 3xH + 2xC + 1xO + 1xN =57.021455 amu

81. Amino Acid Residue Masses Glycine57.02147 Alanine71.03712 Serine87.03203 Proline97.05277 Valine99.06842 Threonine101.04768 Cysteine103.00919 Isoleucine113.08407 Leucine113.08407 Asparagine114.04293 Aspartic acid115.02695 Glutamine128.05858 Lysine128.09497 Glutamic acid129.0426 Methionine131.04049 Histidine137.05891 Phenylalanine147.06842 Arginine156.10112 Tyrosine163.06333 Tryptophan186.07932 Monoisotopic Mass

82. Amino Acid Residue Masses Glycine57.0520 Alanine71.0788 Serine87.0782 Proline97.1167 Valine99.1326 Threonine101.1051 Cysteine103.1448 Isoleucine113.1595 Leucine113.1595 Asparagine114.1039 Aspartic acid115.0886 Glutamine128.1308 Lysine128.1742 Glutamic acid129.1155 Methionine131.1986 Histidine137.1412 Phenylalanine147.1766 Arginine156.1876 Tyrosine163.1760 Tryptophan186.2133 Average Mass

83. Preparing a Peptide Mass Fingerprint Database Take a protein sequence database (Swiss-Prot or nr-GenBank) Determine cleavage sites and identify resulting peptides for each protein entry Calculate the mass (M+H) for each peptide Sort the masses from lowest to highest Have a pointer for each calculated mass to each protein accession number in databank

84. Building A PMF Database >P12345 acedfhsakdfqea sdfpkivtmeeewe ndadnfekqwfe >P21234 acekdfhsadfqea sdfpkivtmeeewe nkdadnfeqwfe >P89212 acedfhsadfqeka sdfpkivtmeeewe ndakdnfeqwfe Sequence DBCalc. Tryptic Frags Mass List acedfhsak dfgeasdfpk ivtmeeewendadnfek gwfe acek dfhsadfgeasdfpk ivtmeeewenk dadnfeqwfe acedfhsadfgek asdfpk ivtmeeewendak dnfegwfe 450.2017 (P21234) 609.2667 (P12345) 664.3300 (P89212) 1007.4251 (P12345) 1114.4416 (P89212) 1183.5266 (P12345) 1300.5116 (P21234) 1407.6462 (P21234) 1526.6211 (P89212) 1593.7101 (P89212) 1740.7501 (P21234) 2098.8909 (P12345)

85. The Fingerprint (PMF) Algorithm Take a mass spectrum of a trypsin-cleaved protein (from gel or HPLC peak) Identify as many masses as possible in spectrum (avoid autolysis peaks of trypsin) Compare query masses with database masses and calculate # of matches or matching score (based on length and mass difference) Rank hits and return top scoring entry – this is the protein of interest

86. Query (MALDI) Spectrum 500 1000 1500 2000 2500 698 2098 1199 1007 609 450 2211 (trp) 1940 (trp)

87. Query vs. Database Query Masses Database Mass List Results 450.2017 (P21234) 609.2667 (P12345) 664.3300 (P89212) 1007.4251 (P12345) 1114.4416 (P89212) 1183.5266 (P12345) 1300.5116 (P21234) 1407.6462 (P21234) 1526.6211 (P89212) 1593.7101 (P89212) 1740.7501 (P21234) 2098.8909 (P12345) 450.2201 609.3667 698.3100 1007.5391 1199.4916 2098.9909 2 Unknown masses 1 hit on P21234 3 hits on P12345 Conclude the query protein is P12345

88. Database search theoreticalexperimental Mascot Protein ID PeptIdent (ExPasy) Mascot (Matrix Science) MS-Fit (Prospector; UCSF) ProFound (Proteometrics) MOWSE (HGMP) Human Genome Mapping Project

90. What You Need To Do PMF A list of query masses (as many as possible) Protease(s) used or cleavage reagents Databases to search (SWProt, Organism) Estimated mass and pI of protein spot (opt) Cysteine (or other) modifications Minimum number of hits for significance Mass tolerance (100 ppm = 1000.0 ± 0.1 Da) A PMF website (Prowl, ProFound, Mascot, etc.)

91. PMF on the Web ProFound –http://129.85.19.192/profound_bin/WebProFound.exe MOWSE http://srs.hgmp.mrc.ac.uk/cgi-bin/mowse PeptideSearch http://www.narrador.embl- heidelberg.de/GroupPages/Homepage.html Mascot www.matrixscience.com PeptIdent http://us.expasy.org/tools/peptident.html

92. ProFound

93. ProFound Results

94. MOWSE

95. PeptIdent

96. MASCOT

97. Mascot Scoring The statistics of peptide fragment matching in MS (or PMF) is very similar to the statistics used in BLAST The scoring probability follows an extreme value distribution High scoring segment pairs (in BLAST) are analogous to high scoring mass matches in Mascot Mascot scoring is much more robust than arbitrary match cutoffs (like % ID)

98. EVD is used as an approximation to model the maxima of long (finite) sequences of random variables Extreme Value Distribution it is the limit distribution of the maxima of a sequence of independent and identically distributed random variables. Because of this, the EVD is used as an approximation to model the maxima of long (finite) sequences of random variables. P(x) = 1 - e -e -x Scores greater than 72 are significant

99. MASCOT

100. Mascot/Mowse Scoring The Mascot Score is given as S = -10*Log(P), where P is the probability that the observed match is a random event Try to aim for probabilities where P<0.05 (less than a 5% chance the peptide mass match is random) 72Mascot scores greater than 72 are significant (p<0.05).

101. Advantages of PMF Uses a “robust” & inexpensive form of MS (MALDI) Doesn’t require too much sample optimization Can be done by a moderately skilled operator (don’t need to be an MS expert) Widely supported by web servers Improves as DB’s get larger & instrumentation gets better Very amenable to high throughput robotics (up to 500 samples a day)

102. Limitations With PMF Requires that the protein of interest already be in a sequence database Spurious or missing critical mass peaks always lead to problems Mass resolution/accuracy is critical, best to have <20 ppm mass resolution Generally found to only be about 40% effective in positively identifying gel spots

103. Tandem Mass Spectrometry Purpose is to fragment ions from parent ion to provide structural information about a molecule Also allows mass separation and AA identification of compounds in complex mixtures Uses two or more mass analyzers/filters separated by a collision cell filled with Argon or Xenon Collision cell is where selected ions are sent for further fragmentation

104. MS-MS & Proteomics

105. Tandem Mass Spectrometry Different MS-MS configurations –Quadrupole-quadrupole (low energy) –Magnetic sector-quadrupole (high) –Quadrupole-time-of-flight (low energy) –Time-of-flight-time-of-flight (low energy)

106. How Tandem MS sequencing works Use Tandem MS: two mass analyzers in series with a collision cell in between Collision cell: a region where the ions collide with a gas (He, Ne, Ar) resulting in fragmentation of the ion Fragmentation of the peptides occur in a predictable fashion, mainly at the peptide bonds The resulting daughter ions have masses that are consistent with known molecular weights of dipeptides, tripeptides, tetrapeptides… Ser-Glu-Leu-Ile-Arg-Trp Collision Cell Ser-Glu-Leu-Ile-Arg Ser-Glu-Leu Ser-Glu-Leu-Ile Etc…

109. Data Analysis Limitations -You are dependent on well annotated genome databases -Data is noisy. The spectra are not always perfect. Often requires manual determination. -Database searches only give scores. So if you have a false positive, you will have to manually validate them

110. Advantages of Tandem Mass Spec FAST No Gels Determines MW and AA sequence Can be used on complex mixtures-including low copy # Can detect post-translational modif.-ICAT High-thoughput capability Disadvantages of Tandem Mass Spec Very expensive-Campus Hardware: $1000 Setup: $300 1 run: $1000 Requires sequence databases for analysis

111. MS-MS & Proteomics Provides precise sequence-specific data More informative than PMF methods (>90%) Can be used for de- novo sequencing (not entirely dependent on databases) Can be used to ID post- trans. modifications Requires more handling, refinement and sample manipulation Requires more expensive and complicated equipment Requires high level expertise Slower, not generally high throughput Advantages Disadvantages

112. ISOTOPE-CODED AFFINITY TAG (ICAT): a quantitative method Label protein samples with heavy and light reagent Reagent contains affinity tag and heavy or light isotopes Chemically reactive group: forms a covalent bond to the protein or peptide Isotope-labeled linker: heavy or light, depending on which isotope is used Affinity tag: enables the protein or peptide bearing an ICAT to be isolated by affinity chromatography in a single step

113. Example of an ICAT Reagent Biotin Affinity tag: Binds tightly to streptavidin-agarose resin Linker: Heavy version will have deuteriums at * Light version will have hydrogens at * Reactive group: Thiol-reactive group will bind to Cys

114. The ICAT Reagent

115. How ICAT works? Proteolysis (ie trypsin) Lyse & Label MIX Affinity isolation on streptavidin beads Quantification MS Identification MS/MS 100 m/z 200400 600 0 100 550570 590 0 m/z Light Heavy NH 2 -EACDPLR- COOH

116. ICAT Quantitation

117. ICAT Advantages vs. Disadvantages Estimates relative protein levels between samples with a reasonable level of accuracy (within 10%) Can be used on complex mixtures of proteins Cys-specific label reduces sample complexity Peptides can be sequenced directly if tandem MS-MS is used Yield and non specificity Slight chromatography differences Expensive Tag fragmentation Meaning of relative quantification information No presence of cysteine residues or not accessible by ICAT reagent

118. Mass Filter Mass Spectrometer Schematic Inlet Ion Source Detector Data System High Vacuum System Turbo pumps Diffusion pumps Rough pumps Rotary pumps Sample Plate Target HPLC GC Solids probe MALDI ESI IonSpray FAB LSIMS EI/CI TOF Quadrupole Ion Trap Mag. Sector FTMS Microch plate Electron Mult. Hybrid Detec. PC’s UNIX Mac

119. MS Detectors Early detectors used photographic film Today’s detectors (ion channel and electron multipliers) produce electronic signals via 2 o electronic emission when struck by an ion Timing mechanisms integrate these signals with scanning voltages to allow the instrument to report which m/z has struck the detector Need constant and regular calibration

120. Mass Detectors Electron Multiplier (Dynode)

121. Limitations of Proteomics -solubility of indiv. protein differs -2D gels unable to resolve all proteins at a given time -most proteins are not abundant (ie kinases) -proteins not in the database cannot be identified -multiple runs can be expensive -proteins are fragile and can be degraded easily -proteins exist in multiple isoforms -no protein equivalent of PCR exists for amplification of small samples

122. Shotgun Proteomics: Multidimensional Protein Identification Technology (MudPIT) Identification Technology (MudPIT)

123. Fractionation & Isolation Peptides Mass Spectrometry Database Search 2-DE Liquid Chromatography Characterization Identification Post Translational modifications Quantification MALDI-TOF MS  -(LC)-ESI-MS/MS General Strategy for Proteomics Characterization

124. SEQUEST ® DTASelect & Contrast Tandem Mass Spectrometer Digestion SCX RP 2D Chromatography MS/MS Spectrum Protein Mixture Peptide Mixture > 1,000 Proteins Identified Overview of Shotgun Proteomics: MudPIT

125. MudPIT Trypsin + proteins p53 IEX-HPLCRP-HPLC

126. MudPIT Cycle  load sample  wash  salt step  wash  RP gradient  re-equilibration x 3~18 SCX RP 2D Chromatography Acquiring MS/MS Datasets Tandem MS Spectrum Peptide Sequence is Inferred from Fragment ions

127. MS/MS of Peptide Mixtures LC MS (MW Profile) MS/MS (AA Identity)

128. Matching MS/MS Spectra to Peptide Sequences CALCULATE COMPARE SCORE SEQUEST ® #1K.TVLIMELINNVAK.K #2 L.NAKMELLIDLVKA.Q #3 E.ELAILMQNNIIGE.N #4 A.CGPSRQNLLNAMP.S #5 L.FAPLQEIINGILE.G … Experimental MS/MS Spectrum Peptides Matching Precursor Ion Mass SEQUEST Output File Theoretical MS/MS Spectra

129. SEQUEST-PVM Beowolf computing cluster 55 mixed CPU: Alpha chips and AMD Athlon PC CPU

130. COMPARE FILTER ASSEMBLE DTASelect Contrast Summary Table ControlA BC PARSE VISUALLY ASSESS SPECTRUM/PEPTIDE MATCHES 20,000s of SEQUEST Output Files Criteria Sets Protein List Filtering, Assembling & Comparing Protein Lists

131. Post Analysis Software DTASelect: Swimming or Drowning in Data It processes tens of thousands of SEQUEST outputs in a few minutes. It applies criteria uniformly and therefore is unbiased. It is highly adaptable and re-analysis with a new set of criteria is easy. It saves time and effort for manual validation. The ‘CONTRAST’ feature can compare results from different experiments.

133. Total Protein Characterization Purification Multiprotein Complex/ Organelle Cells/Tissues Application of shotgun proteomics: Comprehensive Analysis of Complex Protein Mixtures

134. Complete genome sequence information An extensively studied organism Optimal numbers of ORFs, easy for database search Database ORF Unknown, uncoding, hypothetical Known, biochem. or genetics MIPS 6368 1568 4344 YPD 6145 1833 4270 SGD~6000 NA Yeast: A Perfect Model

135. Washburn et al. Nature Biotechnology 19, 242-7 (2001) Functional Categories of Yeast Proteins Identified Ionic Homeostasis Communication and Signal Transduction Cell Rescue, Defense, Death, and Ageing Energy Protein Destination Transcription Transport Protein Synthesis Cell Growth, Division, DNA synthesis, and Biogenesis Unclassified Metabolism Cellular Organization Used GO to determine functional groups

136. Summary of MudPIT It is an automated and high throughput technology. It is a totally unbias method for protein identification. It identifies proteins missed by gel-based methods (i.e. (low abundance, membrane proteins etc.) Post translational modification information of proteins can be obtained, thus allowing their functional activities to be derived or inferred.

137. 2-DE vs MudPIT Widely used, highly commercialized High resolving power Visual presentation Limited dynamic range Only good for highly soluble and high abundance proteins Large amount of sample required Highly automated process low abundance and those from membraneIdentified proteins with extreme pI values, low abundance and those from membrane Thousands of proteins can be identified Not yet commercialized Expensive Computationally intensive Quantitation

138. Peptide Masses From ESI m/z m/z = (MW + nH+) n m/z m/z = mass-to-charge ratio of each peak on spectrum MW = MW of parent molecule n = number of charges (integer) H+ = mass of hydrogen ion (1.008 Da) Each peak is given by:

139. Peptide Masses From ESI 1431.6 = (MW + nH+) n Charge (n) is unknown, Key is to determine MW Choose any two peaks separated by 1 charge 1301.4 = (MW + [n+1]H+) [n+1] 2 equations with 2 unknowns - solve for n first n = 1300.4/130.2 = 10 Substitute 10 into first equation - solve for MW MW = 14316 - (10x1.008) = 14305.914,305.14

140. ESI Transformation Software can be used to convert these multiplet spectra into single (zero charge) profiles which gives MW directly This makes MS interpretation much easier and it greatly increases signal to noise Two methods are available –Transformation (requires prior peak ID) –Maximum Entropy (no peak ID required)

141. Maximum Entropy

142. ESI and Protein Structure ESI spectra are actually quite sensitive to the conformation of the protein Folded, ligated or complexed proteins tend to display non-gaussian peak distributions, with few observable peaks weighted toward higher m/z values Denatured or open form proteins/peptides which ionize easier tend to display many peaks with a classic gaussian distribution

143. ESI and Protein Conformation Native Azurin Denatured Azurin

145. Different MS-MS Modes Product or Daughter Ion Scanning –first analyzer selects ion for further fragmentation –most often used for peptide sequencing Precursor or Parent Ion Scanning –no first filtering, used for glycosylation studies Neutral Loss Scanning –selects for ions of one chemical type (COOH, OH) Selected/Multiple Reaction Monitoring –selects for known, well characterized ions only

146. THE END