Mass spectrometry basics
General scheme of Proteomic analysis                    Protein Mixture              Digestion              Separation Pep...
Information from Mass spectrometryDetermination of Molecular weight.Accuracy of 0.01% of the total molecular weight.Cha...
Uses of mass spectrometerUsed in industry and academia for both routine and researchpurposes. Brief summary of the major ...
Mass spectrometry for biochemistsAccurate molecular weight measurements:confirmation of sample, determination of purity ...
MS divided into 3 fundamental parts                          Mass spectrometer                             Data systemIoni...
Types of Mass spectrometry
Working of Mass Spectrometry Divided into three fundamental parts:Ionization source Analyzer DetectorSample is introd...
Mass spectrometry IntroductionSample                                            Count ions                                ...
Sample introductionThe sample can be inserted directly into the ionization source, or can undergo some   type of chromato...
Matrix-Assisted Laser Desorption/Ionization                      (MALDI)Used for nonvolatile & high Molecular analytesbi...
MALDI PrincipleA laser pulse is used for excitationUV lasers cause electronic excitation IR lasers cause vibrational ex...
MatricesSolidco-crystallization of analyte and matrixoccasionally frozen solventsLiquidIR or UV absorbing liquid cont...
Common Matricesα-cyno-4-hydroxycinnamic acid                                  3-amino-4-hydroxybenzoic acid2.5-dihydrox...
FragmentationMALDI usually gives ‘molecular ions’most often as protonated molecules Control of fragmentationdifference...
Ionization in MALDIIonization is separated into two divisions primary ion formationinitial ions formed during laser puls...
Overview of Mass Spectrometry  Sample Molecule (M)     Ionization          M+/Fragmentation                               ...
Matrix Assisted Laser Desorption IonisationMatrix Assisted Laser Desorption Ionization (MALDI) (F. Hillenkamp, M. Karas, ...
"Somehow, a peak seems to have appeared."Tanaka reported at the weekly Monday teammeeting on February 2nd, 1985, half a ye...
Schematic of a MALDI-TOF Experiment                                  4.Ions are accelerated by an electric                ...
Diagrammatic representation
Laser induced ionization
MALDI-TOF Schematic         optics     Ultraviolet laser                                        Data analysisSample probe ...
Flow chart             Sample dissolved in an appropriate volatile solventAn aliquot of this removed and mixed with a so...
Theoretical Basis of TOF SeparationsFor a particle of Mass = m and charge = z, accelerated through apotential V between p...
Theoretical Basis for TOF-MSCharge = z                zV = ½ mv2Accelerating voltage =    V = ½ mv2/zV                ...
Molecular weight Determination     Molecular Ion     [Mṇ ]⁺                     Dimer                     [Mṇ Mṃ ]⁺       ...
Sample ionization methodsThe ionisation methods used for the majority of biochemical analysesare Electrospray Ionisation...
Ion source   mass analyzer   detector
Electrospray IonizationInvolves transfer of molecules into a vacuum without decomposingthem.Discovered- in the late 80s ...
Electrospray Ionization (ESI) The sample solution is sprayed across a high potential difference (a few  kilovolts) from a...
Electron Spray Ionization(ESI)                 (J. Fenn, J. Phys. Chem., 1984, 88, 4451)Polar molecule analysis, molecules...
Diagrammatic of Electronspray Ionization
Mechanism of Electronspray ionization                                               +     + + - +                         ...
The micro droplet shrinks due tosolvent evaporation The resultingincrease in charge density of thedroplet, forces the cha...
Sample introductionFlow injectionLC/MSTypical flow rates are less than 1 micro literper minute up to about a milliliter...
BenefitsGood for charged, polar or basic compoundsPermits the detection of high-mass compounds at mass-to-charge ratios ...
Limitations Multiply charged species require interpretation and mathematical  transformation (can be difficult sometimes)...
Positive and negative Ion modeIn positive ionization mode, a trace of formic acid is often added to aidprotonation of the...
 In ESI, samples (M) up to 1200 Da give rise to singly charged molecular-  related ions, usually protonated molecular ion...
Expression of m/z value          m/z = (MW + nH+)/ nwhere m/z = the mass-to-charge ratio marked on the abscissa of the spe...
M/Z Spectrum in positive ionization modeLeucine enkephalin         Platform II, BMB, University of Leeds   4 Oct 199910:12...
InterpretationThe m/z spectrum also contains ions at m/z 578.1, some 23 Da higher than theexpected molecular weight. Thes...
Advantages of Multiple Charging Can use instruments with lower maximum m/z (i.e.,  Quadrupoles, ion traps, FTMS) For FTM...
M/Z value expressionLYSO1A 1(1.392)Sm(SG, 2X1.00): Sb(10.10.00)   Platform II, BMB, University of Leeds                   ...
M/Z value expression              m/z = (MW + nH+)                      nwhere  m/z = the mass-to-charge ratio marked on t...
1431.6 = (MW + nH+) and 1301.4 = (MW + (n+1)H+)              n                              (n+1)   These simultaneous equ...
1431.6 = (MW + nH+)                            n   gives                    1431.6 x 10   =     MW + (10 x1.008)   and so ...
Peptide Mass Fingerprinting
peptidesPeptide mass fingerprint              Peptide fragments   Peptide mass search                   MS/MS search
Schematic representation of MALDI-  TOF mass spectrometerMALDI ionization process        MALDI-TOF operating in linear mod...
Peptide Mass Fingerprinting
Effect of Mass accuracy and Mass Tolerance onpeptide Mass Fingerprinting search result       Search m/z    Mass tolerance ...
Effect of Multiple Peptide Masses on ProteinIdentification       Search m/z         Mass tolerance     #Hits       1529.73...
Protein matches for peptide Mass Fingerprintingof m/z 1529.73     Peptide sequence   identification          Matched m/z  ...
Interpretation of PMFExercise for participants
Insilico digested N-terminal peptide for FILGRASTRIM                  N-terminal Peptide Identified                       ...
MTPLGPASSLPQFLIKCLE   MTPLGPASSLPQFLIKCLEN-terminal peptide sequenceN-terminal peptide sequence        m/z=1095         m/...
MTPLGPASSLPQFLIKCLE    MTPLGPASSLPQFLIKCLEAbsence of N-terminal sequence Absence of N-terminal sequence          m/z=1095 ...
Schematic of Triple-Quad MS
Mass Changes from Post translational Modification       Mass Change       Modification       -2.0              Disulfide b...
Ms basics
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Ms basics

  1. 1. Mass spectrometry basics
  2. 2. General scheme of Proteomic analysis Protein Mixture Digestion Separation Peptide mixture Proteins Separation Digestion Peptides MS Analysis M S Data Data Reduction Algorithm Identification
  3. 3. Information from Mass spectrometryDetermination of Molecular weight.Accuracy of 0.01% of the total molecular weight.Changes in mass can be detected e.g. substitution ofone amino acid for another, or a post-translationalmodification.Structural information can be achieved by fragmentingthe sample and analyzing the products generated.
  4. 4. Uses of mass spectrometerUsed in industry and academia for both routine and researchpurposes. Brief summary of the major mass spectrometricapplications: •Biotechnology: the analysis of proteins, peptides, oligonucleotides •Pharmaceutical: drug discovery, combinatorial chemistry, pharmacokinetics, drug metabolism •Clinical: neonatal screening, hemoglobin analysis, drug testing •Environmental: PAHs, PCBs, water quality, food contamination •Geological: oil composition
  5. 5. Mass spectrometry for biochemistsAccurate molecular weight measurements:confirmation of sample, determination of purity of a sample, verifying amino acidsubstitutions, detection of post-translational modifications, calculating number ofdisulphide bridges . Reaction monitoring:to monitor enzyme reactions, chemical modification, protein digestion Amino acid sequencing:sequence confirmation, de novo characterisation of peptides, identification ofproteins by database searching with a sequence “tag” from a proteolytic fragment Oligonucleotide sequencing:the characterization or quality control of oligonucleotides Protein structure:protein folding monitored by H/D exchange, protein-ligand complex formationunder physiological conditions, macromolecular structure determination
  6. 6. MS divided into 3 fundamental parts Mass spectrometer Data systemIonization source Analyser Detectore.g. electrospray(ESI), Mass to charge,m/z e.g. photomultiplier,Matrix assisted laser e.g. quadrupole, Microchannel plate,Desorption(MALDI) Time of flight, Electron multiplier magnet, FT-ICR
  7. 7. Types of Mass spectrometry
  8. 8. Working of Mass Spectrometry Divided into three fundamental parts:Ionization source Analyzer DetectorSample is introduced in the ionization source where they are ionized. ( It is easier to manipulate ions than neutral molecules).Ions separated according to their mass to charge ratio in the analyzer.The separated ions are detected and this signal sent to a data systemwhere the m/z ratios are stored together with their relative abundancefor presentation in the format of a m/z spectrum. The separated ions are detected and this signal sent to a data systemwhere the m/z ratios are stored together with their relative abundancefor presentation in the format of a m/z spectrum.
  9. 9. Mass spectrometry IntroductionSample Count ions Separateintroduction Collect results masses Ionization Minimize collisions, interferences
  10. 10. Sample introductionThe sample can be inserted directly into the ionization source, or can undergo some type of chromatography en route to the ionisation source. Gas source (lighter elements) dual inlet - sample purified and measured with standard gas at identical conditions precisions ~ ±0.005% continuous flow - sample volatized and purified (by EA or GC) and injected into mass spec in He carrier gas, standards measured before and after, precisions ~ 0.005-0.01% Solid source (heavier elements) TIMS - sample loaded onto Re filament, heated to ~1500°C, precisions ~0.001% laser ablation - sample surface sealed under vacuum, then sputtered with laser precisions ~0.01%Inductively coupled plasma (all elements) ICPMS - sample converted to liquid form, converted to fine aerosol in nebulizer, injected into ~5000K plasma torch
  11. 11. Matrix-Assisted Laser Desorption/Ionization (MALDI)Used for nonvolatile & high Molecular analytesbiopolymers and oligomers proteins, peptides, oligonucleotides, oligosaccharidessynthetic polymersInorganic, such a fullerenes environmental compounds kerogens, coal tars, humic acids, fulvic acids
  12. 12. MALDI PrincipleA laser pulse is used for excitationUV lasers cause electronic excitation IR lasers cause vibrational excitationMatrix molecules transfer energy100-50,000 x [A]low analyte fragmentationmatrices are selectableTOF-MS is used for analysis
  13. 13. MatricesSolidco-crystallization of analyte and matrixoccasionally frozen solventsLiquidIR or UV absorbing liquid containing analyteTwo phase liquids with absorbing solid
  14. 14. Common Matricesα-cyno-4-hydroxycinnamic acid 3-amino-4-hydroxybenzoic acid2.5-dihydroxybenzoic acid Sinapinic acid
  15. 15. FragmentationMALDI usually gives ‘molecular ions’most often as protonated molecules Control of fragmentationdifferences in sublimation temperature of matrices control thermalexcitation PA and IP of a matrix affect energy transfer involved withprotonation and electron transfercollisionsextraction through neutral plumeresidual gas
  16. 16. Ionization in MALDIIonization is separated into two divisions primary ion formationinitial ions formed during laser pulsefrequently matrix moleculessecondary ion formationions formed during subsequent reactionsmay be matrix-matrix reactions or matrix-analyte reactionsResulting analyte ions are usuallyprotonatedCationized radical cations
  17. 17. Overview of Mass Spectrometry Sample Molecule (M) Ionization M+/Fragmentation Mass Analyzer Mass Spectrum Protonation : M + H+ MH Cationization : M + Cat+ MCat+Mechanism of Ionization Deprotonation: MH M - + H+ Electron Ejection: M M+. + e- Electron Capture: M + e- M-.
  18. 18. Matrix Assisted Laser Desorption IonisationMatrix Assisted Laser Desorption Ionization (MALDI) (F. Hillenkamp, M. Karas, R. C.Beavis, B. T. Chait, Anal. Chem., 1991, 63, 1193), deals well with thermolabile, non-volatile organic compounds for the analysis of proteins, peptides, glycoprotein,oligosaccharides, and oligonucleotides.Measures masses within 0.01% of the molecular weight of the sample, at least upto ca. 40,000 Da. Based on the bombardment of sample molecules with a laser light to bring aboutionisation. Sample is pre-mixed with absorbing matrix compound for the most reliableresults, and a low concentration of sample to matrix works best.Matrix transforms the laser energy into excitation energy for the sample, leads tosputtering of analyte and matrix ions from the surface of the mixture.In this way energy transfer is efficient and also the analyte molecules are sparedexcessive direct energy that may otherwise cause decomposition.Most commercially available MALDI mass spectrometers now have a pulsednitrogen laser of wavelength 337 nm.
  19. 19. "Somehow, a peak seems to have appeared."Tanaka reported at the weekly Monday teammeeting on February 2nd, 1985, half a yearafter the project had started. Six Oclock in the Evening on October 9th 2002 News arrived saying that Koichi Tanaka had won the Nobel Prize in Chemistry 2002 On October 9th, the Royal Swedish Academy of Sciences announced their decision to award the Nobel Prize in Chemistry 2002 to three people for their development of methods for identification and structure analyses of biological macromolecules. - Koichi Tanaka (at the time : Life Science Laboratory Assistant Manager of Shimadzu Corporation), Prof. John B. Fenn (Virginia Commonwealth University, USA) and Prof. Kurt Wuthrich (Swiss Federal Institute of Technology)
  20. 20. Schematic of a MALDI-TOF Experiment 4.Ions are accelerated by an electric field to the same kinetic energy and1. Sample is they drift down the field free flight mixed in tube where they are separated in matrix and space dried on target. 5.Ions strike the2.Target is introduced detector at different into high vacuum of times depending on MS. the mass to charge ratio of the ions 3.Sample is irradiated with laser desorbing ions into the gas phase and the clock measuring the time of 6.A data system controls all flight starts. the parameters, acquires the signal vs. time and permits data processing
  21. 21. Diagrammatic representation
  22. 22. Laser induced ionization
  23. 23. MALDI-TOF Schematic optics Ultraviolet laser Data analysisSample probe oscilloscope amplifiervacuum Deflection plates
  24. 24. Flow chart Sample dissolved in an appropriate volatile solventAn aliquot of this removed and mixed with a solution containing a vast excess ofa matrix. sinapinic acid( protein analysis), ±-cyano-4-hydroxycinnamicacid(peptide analysis)An aliquot applied to the sample, allowed to dry prior to insertion into the highvacuum. laser is fired, the energy arriving at the sample/matrix surface optimized,and data accumulated as m/z spectrumTof analyzer separates ions according to their m/z ratios. The heavier ions areslower than the lighter ones.Results in the generation of singly charged ions regardless of the molecular weight. In +ve ionisation mode the protonated molecular ions (M+H+) are usually thedominant species. Positive ionisation is used in general for protein and peptideanalyses.In -ve ionisation mode the deprotonated molecular ions (M-H-) are usually the mostabundant species. Negative ionisation can be used for the analysis of oligonucleotidesand oligosaccharides.
  25. 25. Theoretical Basis of TOF SeparationsFor a particle of Mass = m and charge = z, accelerated through apotential V between plates distance d apart:How long t does it take to complete the trip?What is final speed v?What is the relationship between t (time-of flight) andM/z (mass to charge ratio)?
  26. 26. Theoretical Basis for TOF-MSCharge = z zV = ½ mv2Accelerating voltage = V = ½ mv2/zV 2V= mv2/zMass = m but v = d/tVelocity = v m/z = [2V/d2]t2Distance = d t = (m/z)1/2(d2/V)1/2TOF = t
  27. 27. Molecular weight Determination Molecular Ion [Mṇ ]⁺ Dimer [Mṇ Mṃ ]⁺ Trimer Tetramer
  28. 28. Sample ionization methodsThe ionisation methods used for the majority of biochemical analysesare Electrospray Ionisation (ESI)Matrix Assisted Laser Desorption Ionisation (MALDI). Other Ionisation methods include: Atmospheric Pressure Chemical Ionisation (APCI) Chemical Ionisation (CI) Electron Impact (EI) Fast Atom Bombardment (FAB) Field Desorption / Field Ionisation (FD/FI) Thermo spray Ionisation (TSP)
  29. 29. Ion source mass analyzer detector
  30. 30. Electrospray IonizationInvolves transfer of molecules into a vacuum without decomposingthem.Discovered- in the late 80s in the group of Prof. Fenn at Yale.The intact transfer of large molecules from the liquid gasphase by an ion desorption mechanism, a direct emission of largemolecules from liquid droplets.Operate under atmospheric conditions.
  31. 31. Electrospray Ionization (ESI) The sample solution is sprayed across a high potential difference (a few kilovolts) from a needle into an orifice in the interface. Heat and gas flows are used to desolvate the ions existing in the sample solution. Electrospray ionization can produce multiply charged ions with the number of charges tending to increase as the molecular weight increases. The number of charges on a given ionic species must be determined by methods such as: comparing two charge states that differ by one charge and solving simultaneous equations looking for species that have the same charge but different adduct masses examining the mass-to-charge ratios for resolved isotopic clusters
  32. 32. Electron Spray Ionization(ESI) (J. Fenn, J. Phys. Chem., 1984, 88, 4451)Polar molecule analysis, molecules ranging from less than 100 Da to more than1,000,000 Da in molecular weight. ProcedureSample is dissolved in a polar, volatile solvent and pumped through a narrow,stainless steel capillary. High voltage applied to the tip of the capillary.Sample emerging from the tip is dispersed into an aerosol of highly chargeddroplets, aided by nebulising gas flowing around the outside of the capillary.Charged droplets diminish in size by solvent evaporation, assisted by a warmflow of nitrogen known as the drying gas which passes across the front of theionisation source.Charged sample ions, free from solvent, are released from the droplets, passthrough an orifice into an intermediate vacuum region, and from there intothe analyzer of the mass spectrometer, under high vacuum.
  33. 33. Diagrammatic of Electronspray Ionization
  34. 34. Mechanism of Electronspray ionization + + + - + + + - + - + - + + + + ++- - + - In volatile Solvent evaporates As fieldOriginal droplet Field increases, and increases, residueContains + and – Ions move toward ions areIons; + surface emitted from predominant drop
  35. 35. The micro droplet shrinks due tosolvent evaporation The resultingincrease in charge density of thedroplet, forces the chargedanalyte ion out of the solutionbefore the droplet breaks up.
  36. 36. Sample introductionFlow injectionLC/MSTypical flow rates are less than 1 micro literper minute up to about a milliliter perminute.
  37. 37. BenefitsGood for charged, polar or basic compoundsPermits the detection of high-mass compounds at mass-to-charge ratios that are easily determined by most massspectrometers (m/z typically less than 2000 to 3000).Best method for analyzing multiply charged compounds.Very low chemical background leads to excellent detectionlimits.Can control presence or absence of fragmentation bycontrolling the interface lens potentials.Compatible with MS/MS methods.
  38. 38. Limitations Multiply charged species require interpretation and mathematical transformation (can be difficult sometimes). Complementary to APCI. Not good for uncharged, non-basic, low- polarity compounds (e.g. steroids). Very sensitive to contaminants such as alkali metals or basic compounds. Relatively low ion currents Relatively complex hardware compared to other ion sources Mass range Low-high Typically less than 200,000 Da.
  39. 39. Positive and negative Ion modeIn positive ionization mode, a trace of formic acid is often added to aidprotonation of the sample molecules.In negative ionization mode a trace of ammonia solution or a volatile amine isadded to aid deprotonation of the sample molecules.Proteins and peptides are usually analyzed under positive ionisation conditionsand saccharides and oligonucleotides under negative ionisation conditions.In all cases, the m/z scale must be calibrated by analyzing a standard sample.Positive or Negative Ionizsation? If the sample has functional groups that readily accept a proton (H+) then positive ion detection is used e.g. amines R-NH2 + H+ ® R-NH3+ as in proteins, peptides If the sample has functional groups that readily lose a proton then negative ion detection is used e.g. carboxylic acids R-CO2H ® R-CO2– and alcohols R-OH ® R- O– as in saccharides, oligonucleotides
  40. 40.  In ESI, samples (M) up to 1200 Da give rise to singly charged molecular- related ions, usually protonated molecular ions of the formula (M+H)+ in positive ionisation mode and deprotonated molecular ions of the formula (M-H)- in negative ionisation mode. Samples (M) with molecular weights > 1200 Da give rise to multiply charged molecular-related ions such as (M+nH)n+ in positive ionisation mode and (M-nH)n- in negative ionisation mode. Proteins have many suitable sites for protonation as all of the backbone amide nitrogen atoms could be protonated theoretically, as well as certain amino acid side chains such as lysine and arginine which contain primary amine functionalities.
  41. 41. Expression of m/z value m/z = (MW + nH+)/ nwhere m/z = the mass-to-charge ratio marked on the abscissa of the spectrum; MW = the molecular weight of the sample n = the integer number of charges on the ions H = the mass of a proton = 1.008 Da.
  42. 42. M/Z Spectrum in positive ionization modeLeucine enkephalin Platform II, BMB, University of Leeds 4 Oct 199910:12:26 TEST0132(1.679)cm(3:34) Scan ES+ 2.87 e5 ESI-MS analysis of Leucine enkephalin Calculated MW.555.2 Da Measured MW.555.1Da
  43. 43. InterpretationThe m/z spectrum also contains ions at m/z 578.1, some 23 Da higher than theexpected molecular weight. These can be identified as the sodium adduct ions,(M+Na)+, and are quite common in electrospray ionization.Electrospray ionization is known as a “soft” ionization method as the sampleis ionised by the addition or removal of a proton, with very little extra energyremaining to cause fragmentation of the sample ionsBy raising the voltage applied to the sampling cone, extra energy issupplied to the sample ions which can then fragment. The m/z spectrumthen has extra peaks corresponding to sample fragment ions which can helpin the structural elucidation of the sample.  Known as “cone voltage” or “in-source” fragmentation and although it can provide useful information it must be remembered that it is not specific so if there are a number of components in a sample, all will fragment to give rise to an extremely complicated spectrum.
  44. 44. Advantages of Multiple Charging Can use instruments with lower maximum m/z (i.e., Quadrupoles, ion traps, FTMS) For FTMS, the resolution is better at lower m/z values, therefore, ESI helps one obtain better resolution at higher m/z values. Multiply charge ions tend to fragment easier then singly charge ions.
  45. 45. M/Z value expressionLYSO1A 1(1.392)Sm(SG, 2X1.00): Sb(10.10.00) Platform II, BMB, University of Leeds 25 Jan 2000 10:10:37 Hen egg Lysozyme ScanES+ 2.16e6
  46. 46. M/Z value expression m/z = (MW + nH+) nwhere m/z = the mass-to-charge ratio marked on the abscissa of the spectrum; MW = the molecular weight of the sample n = the integer number of charges on the ions H = the mass of a proton = 1.008 Da.
  47. 47. 1431.6 = (MW + nH+) and 1301.4 = (MW + (n+1)H+) n (n+1) These simultaneous equations can be rearranged to exclude the MW term: n(1431.6) –nH+ = (n+1)1301.4 – (n+1)H+and so n(1431.6) = n(1301.4) +1301.4 – H+ therefore: n(1431.6-1301.4)= 1301.4 – H+ and: n= (1301.4 - H+) (1431.6 – 1301.4) hence the number of charges on the ions at m/z 1431.6 = 1300.4 = 10. 130.2
  48. 48. 1431.6 = (MW + nH+) n gives 1431.6 x 10 = MW + (10 x1.008) and so MW = 14,316 – 10.08 therefore MW = 14,305.9 Da
  49. 49. Peptide Mass Fingerprinting
  50. 50. peptidesPeptide mass fingerprint Peptide fragments Peptide mass search MS/MS search
  51. 51. Schematic representation of MALDI- TOF mass spectrometerMALDI ionization process MALDI-TOF operating in linear mode MALDI-TOF instrument equipped with a reflection
  52. 52. Peptide Mass Fingerprinting
  53. 53. Effect of Mass accuracy and Mass Tolerance onpeptide Mass Fingerprinting search result Search m/z Mass tolerance #Hits 1529 1 478 1529.7 0.1 164 1529.73 0.01 25 1529.734 0.001 4 1529.7348 0.0001 2 Searches were done with the MS-FIT program
  54. 54. Effect of Multiple Peptide Masses on ProteinIdentification Search m/z Mass tolerance #Hits 1529.73 0.1 204 1529.73 1529.7O 0.1 7 1529.73 1252.7O 1833.88 0.1 1 Searches were done with the MS-FIT program The Actual Peptide M/Z values are 1529.7348,1252.7074,1833.8845
  55. 55. Protein matches for peptide Mass Fingerprintingof m/z 1529.73 Peptide sequence identification Matched m/z IGGHGAEYGAEALER Mouse Hb alpha 1529.7348 VGAHAGEYGAEALER Human Hb alpha 1529.7348 MGTGWEGMYRTLK Mouse lens epithelial 1529.7245 Cell protein LEP503 MADEEKLPPGWEK Human PINI-like 1529.7310 protein DTQTSITDSSAIYK Mouse signal 1529.7335 recognition particle NDSSPNPVYQPPSK Mouse peroxisome 1529.7236 assembly factor-1 MNLSLNDAYDFVK Human dual 1529.7310 specificity protein phosphatase 7
  56. 56. Interpretation of PMFExercise for participants
  57. 57. Insilico digested N-terminal peptide for FILGRASTRIM N-terminal Peptide Identified + + Carbamidomethylation 57 DaTotal 2188 Da
  58. 58. MTPLGPASSLPQFLIKCLE MTPLGPASSLPQFLIKCLEN-terminal peptide sequenceN-terminal peptide sequence m/z=1095 m/z=1095
  59. 59. MTPLGPASSLPQFLIKCLE MTPLGPASSLPQFLIKCLEAbsence of N-terminal sequence Absence of N-terminal sequence m/z=1095 m/z=1095 MTPLGPASSLPQFLIKCLE MTPLGPASSLPQFLIKCLE N-terminal peptide sequence N-terminal peptide sequence m/z=1095 m/z=1095
  60. 60. Schematic of Triple-Quad MS
  61. 61. Mass Changes from Post translational Modification Mass Change Modification -2.0 Disulfide bond formation +14.0 Methylation +16.0 Hydroxylation +28.0 Formylation +30.0 Nitrosylation +42.0 Acetylation +80.0 Sulfation +80.1 Phosphorylation +180 Mono-glycosylation +204.4 Farnesylation +210.4 Myristoylation

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