Proteomics. George Tsaprailis, Director Linda Breci, Associate Director. Arizona Proteomics Consortium University of Arizona

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1 Proteomics George Tsaprailis, Director Linda Breci, Associate Director Arizona Proteomics Consortium University of Arizona What is proteomics and how is Mass spectrometry used in proteomics? 1

2 Proteomics: the study of the Proteome A collection of proteins, usually comprising a biological system Important because (1) proteins perform most cellular functions, (2) proteins are the major elements of most cellular structures, and (3) proteins are targets of drugs/toxicants Why Proteomics? Mass Spectrometry Protein ID 2

3 Proteomics involves- Protein Chemistry Mass Spectrometry Computing (+ Bioinformatics) Protein Chemistry Sample isolation/clean-up Sample purification fractions Protein digest peptides 3

4 Key to Proteomics is to obtain peptide masses and/or sequences Mass Spectrometry Protein mixture digestion separation Proteins digestion Peptide mixture separation Peptides MS analysis MS data MS MS/MS All types of hardware used in proteomics Mass Spectrometry 4

5 Computing (+ Bioinformatics) The Proteomic Approach Sample Pre-prep steps 1D PAGE 2D PAGE Protein Chemistry Protein(s) Solution HPLC fractions IP eluent Protein ESI Mass Spectrometry LC-MS/MS MALDI MS Mass Spectrometer Digest Peptides Computing (+ Bioinformatics) Protein Id + Informatics 5

6 What is proteomics and how is Mass spectrometry used in proteomics? Mass Spectrometry What is a mass spectrometer and what does it measure? An instrument that makes ions Measures the mass/charge (m/z) of ions Mass Spectrometry in proteomics For proteins and peptides whole protein mass measurements protein identification based on peptide mass measurement protein identification based on peptide structure analysis (fragmentation) Need to know some basic principles 6

7 Protein/peptide relationship Enzyme Protein Peptides Making ions H+ O H 2 N CH C O H N CH C O H N CH C O H N CH C OH CH 3 CH 2 CH 2 CH 2 CH CH 3 CH 2 CH 3 CH 2 H+ CH 2 NH 2 Ala-Leu-Phe-Lys mass of neutral = Ala-Leu-Phe-Lys m/z of singly charged = Ala-Leu-Phe-Lys m/z of doubly charged =

8 Making ions Ions are made in an ion source Important methods in Proteomics: 1) MALDI (matrix assisted laser desorption) 2) ESI (electrospray ionization Electrospray Ionization ESI Calculated Mass Spectrum Relative Intensity Intensity m/z m/z [M+H] Intensity [M+2H] Matrix Assisted Laser Desorption Ionization MALDI 4 2 [2M+H] , m/z 8

9 Analyzing ions The ion source is coupled to the analyzer Important analyzers in Proteomics: 1) TOF (time of flight) 2) Ion Trap Matrix Assisted Laser Desorption (MALDI) (ion source) Time of Flight (analyzer) FLIGHT TUBE ANALYZER Pulsed laser light Analyzer LASER Ion Beam Detector Sample and matrix on tip of solid probe 9

10 Time of Flight (TOF) Linear Mode: better sensitivity poor resolution Reflectron Mode: less sensitivity higher resolution MALDI-TOF spectrum (mix of peptides) 9 x 4. Ref Ref 5 m/z 25 D:\113_5fmol\Bsaintcal\2Ref\pdata\1\1r (11:26 1/4/1) 1

11 MALDI Reflectron Spectrum Of ACTH 13-Nov-23 ACTHResCk 3 (.98) Cm (1:5) 1 M@LDI TOF LD+ 6.57e % m/z Electrospray (ESI) (ion source) Ion Trap (analyzer) ION TRAP ANALYZER ESI HPLC 45 V Dry gas or Heat Analyzer Ion Beam Detector Liquid sample sprayed from needle or capillary 11

12 ESI-Ion Trap Spectrum 1 [M+H] [M+H] + = = [M+2H] 2+ = ( ) / 2 = Relative Intensity 6 4 [M+2H] [2M+H] + = (951.4 x 2) + 1 = [2M+H] m/z Exercise 1 View an ion trap animation 12

13 Resolution and mass accuracy varies by instrument INSTRUMENT MASS RANGE Resolution Accuracy (Error) m/z (at m/z 1,) (at m/z 1,) LCQ (Ion Trap) to 2, 2, (full scan).3% (3 ppm) 1, (zoom scan) MALDI/TOF to 4, 15, (reflectron).6% (6 ppm) Ext. Cal..3% (3ppm) Int.Cal. FTICR to 4, 5,.1% (1ppm) ( TheoreticalMW MeasuredMW) ppm = 1 TheoreticalMW 6 Resolution Resolution 3, 1, 3, 1, 13

14 MALDI Reflectron Spectrum Of ACTH 13-Nov-23 ACTHResCk 3 (.98) Cm (1:5) 1 M@LDI TOF LD+ 6.57e % m/z You must know the resolution of your instrument to analyze the data! We need to know the possible error in the measurement Is the peak monoisotopic? Is the peak average? 14

15 Analysis of whole proteins by MALDI-TOF and ESI-Ion trap MALDI-TOF = measure with 1 or 2 protons large molecules like Proteins require Linear mode (much lower resolution) ESI-Ion Trap = measure with many protons (high charge state) mass of the protein can be calculated from the multiply charged peaks Mass Spec measures isotopes Excel calculated example: Carbon is 12. For every 12 C there is 1.1% 13 C isotopes add up 1 carbons = 11% 13 C Peak for 1 carbons, the 13 C peak is larger than the 12 C peak 1 carbons 1 carbons

16 Proteins have very large isotope widths Theoretical Isotope distribution of Lysozyme 1st Isotope th Isotope Isotope # m /z % Maximum Lysozyme by MALDI/TOF Average mass = 14, [M+H] [M+H] Intensity [2M+2H] [M+2H] [2M+H] m/z 16

17 Lysozyme by ESI-Ion Trap Average mass = 14, Calculated Mass Spectrum Relative Intensity Intensity m/z m/z So we ve made measurements Now What? A lot of information is available on-line about proteins and/or the gene We will explore protein information in general We will then use the available info to perform data analysis 17

18 MALDI-TOF analysis of Alkaline phosphatase Computer Exercise #2 1.2E+9 Intensity 1.E+9 8.E+8 6.E+8 4.E+8 [M+2H] [M+H] [2M+H] E m/z Protein identification Two strategies single stage mass spectrometry (MS) called peptide mass mapping measure all peptides in one spectrum MALDI-TOF produces low confidence results tandem mass spectrometry (MS/MS) measure peptides as they elute from an HPLC ESI-Ion Trap produces high confidence results 18

19 Single Stage Peptide mass mapping Using MALDI-TOF MALDI-TOF Spectrum of tryptic digest 9 x 4. Ref Ref 5 m/z 25 D:\113_5fmol\Bsaintcal\2Ref\pdata\1\1r (11:26 1/4/1) 19

20 MALDI Reflectron Spectrum Of ACTH 13-Nov-23 ACTHResCk 3 (.98) Cm (1:5) 1 M@LDI TOF LD+ 6.57e % m/z Data Analysis for peptide mass mapping MS? protein peptides identify for example: Measured Peptide = rank MS Peptide MW Found in Selected Databases NDALYFPT... SWDLTAL... PTDLDVSY... Important data multiple peaks mass accuracy confirming information (pi, approx. mass, organism, etc.) 2

21 Data Analysis for peptide mass mapping >gi ref NP_ solute carrier family 6 (neurotransmitter transporter, glycine), member 9 [Bos taurus]gi gb AAB glycine transporter MAAAQGPVAPSKLEQNGAVPSEATKSDQNLGQGNWRNQIEFVLTSVGYAVGLGNV WRFPYLCYRNGGGAFMFPYFIMLIFCGIPLFFMELSFGQFASQGCLGVWRISPMFK GVGYGMMVVSTYIGIYYNVVICIAFYYFFSSMTPVLPWTYCNNPWNTPDCMSVLDN PNITNGSQPPALPGNVSQALNQTLKRTSPSEEYWRLYVLKLSDDIGNFGEVRLPLLG CLGVSWVVVFLCLIRGVKSSGKVVYFTATFPYVVLTILFIRGVTLEGAFTGIMYYLTPQ WDKILEAKVWGDAASQIFYSLGCAWGGLVTMASYNKFHNNCYRDSVIISITNCATSV YAGFVIFSILGFMANHLGVDVSRVADHGPGLAFVAYPEALTLLPISPLWSLLFFFMLILL GLGTQFCLLETLVTAIVDEVGNEWILQKKTYVTLGVAVAGFLLGIPLTSQAGIYWLLLM DNYAASFSLVIISCIMCVSIMYIYGHQNYFQDIQMMLGFPPPLFFQICWRFVSPAIIFFIL IFSVIQYQPITYNQYQSSQTGLPLFTCQIAPAHVPQPLSGARTPSPKPWSVRVSVLRA PLCSDSPGRAASNPL MAAAQGPVAPSK = LEQNGAVPSEATK = SDQNLGQGNWR = Measured Peptide = theoretical measured.695 difference error = 55 ppm Data Analysis for peptide mass mapping MS? protein peptides identify MS Peptide MW Found in Selected Databases NDALYFPT... SWDLTAL... PTDLDVSY... Important data multiple peaks mass accuracy confirming information (pi, approx. mass, organism, etc.) rank 21

22 Computer Exercise #4 Analyze peptide mass mapping data 4 lists of peptide masses provided on worksheet (Alternate address of excel data): Problems with whole protein analysis Peaks are broad large groups of isotope peaks peaks further broadened by adducts (contaminants, salts) Proteins are often modified Instrument may not resolve the mass difference No information regarding which amino acid is modified Proteins are in a complex matrix background stuff other proteins (too complex!!!) Therefore proteins are identified from peptides! 22

23 How are proteins separated Proteins from biological organisms are a complex mixture Separating proteins 1D SDS-PAGE Cross linking controls MW separated Low resolution technique, spot can contain 1's to 1's of proteins 2D SDS-PAGE Best for complex protein mixtures (IEF + SDS-PAGE) Other methods Chromatography (reverse phase, size exclusion, ion exchange, affinity) Preparative isolectric focusing (IEF) 1D Electrophoresis Great clean-up tool (rid of salts, detergents, etc ) Great concentration tool Biological analytes Various stains available various detection limits USE PRECAST GELS (polymer issue) if possible Various size gels (spatial resolution) Various MW ranges Protein Mixture or IP eluant 1D SDS-PAGE 23

24 1D Electrophoresis 2D Electrophoresis (1) Separation on the basis of intrinsic charge (pk a ) isoelectric focusing (2) Separation on the basis of Size PAGE (SDS gel electrophoresis) 24

25 2D Electrophoresis Protein Mixture or IP eluant or Cell/tissue 2D SDS-PAGE Great clean-up tool (rid of salts, detergents, etc ) Various stains available various detection limits Protein profiling Various ph ranges 2D gels are very much sample related (sample may require further clean-up prior to 2D gel Avoid excess salts in sample (not focus, IPGs burn, 3-4 mm max salt) Often Automated w/ robotics high throughput (MALDI-TOF) Often good for visualizing PTMs The 1 st D: Isoelectric Focussing + ph 3 ph 7.5 ph 1 + ph 3 ph 7.5 ph 1 + ph 3 ph 7.5 ph 1 + ph 3 ph 7.5 ph 1 25

26 The 2 nd D: SDS-PAGE Proteins migrate through the gel at a rate proportional to their size Smallest proteins travel the furthest distance + + ph 3 ph 3 ph 7.5 ph 7.5 ph1 ph1 ph 3 ph 7.5 ph1 size charge Do Computer Exercise #3 Laboratory tour 26