1、Analysis of Proteins by Mass Spectrometry,Paolo Lecchi Dept. of Pharmacology and Physiology George Washington University,April 12, 2004,DIFFERENTIAL EXPRESSION AND QUANTITATION,THE ROLE OF MASS SPECTROMETRY IN PROTEIN ANALYSIS:,IDENTIFICATION OF PROTEINS (USUALLY IN COMPLEX MIXTURES),STRUCTURAL ANAL
2、YSIS OF PROTEIN CHARACTERIZATION OF PROTEIN MODIFICATION, (e.g., POSTRANSLATIONAL and CHEMICAL),WHAT IS A “MASS SPECTROMETER ”.?,The black box problem.,“A MASS SPECTROMETER MEASURES THE MW.”,“.A MS ANALYSIS GIVES THE MASS-TO-CHARGE RATIO (m/z) OF IONSIN GAS PHASE”.,ESI,Liquid flow,Q or Ion Trap anal
3、yzer,an Electrospray Ionization (ESI) source gives a continuous stream of ions, best for quadrupoles, ion traps, etc.,MALDI,3 nS LASER PULSE,Sample (solid) on target at high voltage/ high vacuum,MALDI (Matrix Assisted Laser Desorption Ionization)ions are generated in a discontinous way best suited t
4、o time-of-flight MS.,TOF analyzer,Atmosphere Low vac. High vac.,High vacuum,.MALDI or Electrospray ?,MALDI is limited to solid state, ESI to liquid,ESI is better for the analysis of complex mixture as it is directly interfaced to a separation techniques (i.e. HPLC or CE),Until recently only ESI was
5、available for high quality tandem-MS,MALDI is easier to use and maintain,MALDI is more “flexible” (MW from 200 to 400,000 Da),A “RESEARCH GRADE” MS (200 to 500 k$) PROVIDES AN ACCURATE MW DETERMINATION: 10 ppm (e.g. 1000.0 0.1),ACCURACY IS NOT THE ONLY PARAMETER TO BE CONSIDERED IN A MASS SPECTROMET
6、ER.,.dont forget the “S factors”,Sensitivity = femtomole 10-15 M (.attomole 10-18 M),Simplicity = very easy training required,$ = 70 to 650 k$ 120 to 650 k$,Speed (high throughput)= 104/day dynamic system,Structural information = MS/MS MSn,Software = “ .evaluation in progress.”,MALDI ESI,Selectivity
7、 (resolution) = 5000,ACTUAL SPECTRUM,ESI-ion trap spectra of apomyoglobin,MALDI-TOF spectra of apomyoglobin,INSTRUMENT: Kratos Axima-CFR,Sample: 1 pmole apomyoglobin (horse skeletal muscle),“.for their developments of soft desorption ionisation methods for mass spectrometric analysis of biological m
8、acromolecules”.,Nobel Prize in Chemistry 2002,1/2 of the prize went to Kurt Wutrich (Switzerland) development of NMR analysis,1/4 to John B. Fenn (USA) Virginia Commonwealth University,1/4 to Koichi Tanaka (Japan) Shimadzu. Corp. Kyoto,Electrospray,Laser Ionization,Our new MALDI TOF : Kratos (Shimad
9、zu) AXIMA-CFR,Run 2Dgel; stain; scan,2D-PAGE AND MASS PECTROMETRY. A PARADIGM IN PROTEOMICS,PROTEOMICS (the analysis of the “proteinaceous” material) is a tremendous analytical challenge,AN IDEAL METHOD SHOULD BE ABLE TO IDENTIFY AND QUANTIFY PROTEINS WHOSE EXPRESSION LEVELS CHANGE,THERE IS A HUGE D
10、YNAMIC RANGE OF PROTEIN EXPRESSION (12 orders of magnitude),MORE THAN OTHER ANALYITICAL TECHNIQUES PROTEOMICS IS AFFECTED BY THE “GIGO” FACTOR,(GARBAGE-IN, GARBAGE-OUT),NO “PCR-EQUIVALENT” METHODS FOR PROTEINS,SEPARATION electrophoresis (1-D, 2-D)chromatography (SEC, ion exchange, reversed phase),DI
11、GESTION chemical (BrCN)enzymatic (trypsin, Lys-C, Asp-C)reduction (Di-Thio-Threitol, b-Mercapto-Ethanol)alkylation (IodoAcAcid, IodoAcAmide, Vynil Pyridine),MALDI MS ANALYSISprotein identification (peptide mass fingerprinting)peptide structural information (post source decay),EXPERIMENTAL PROCEDURES
12、 IN PROTEOMICS,SAMPLE CLEAN UP chromatography (reversed phase),solid phase extraction (Zip Tip),Mass spectrometry improved substantially during the last 10 years.,2D-PAGE still is the most powerful separation technique but has several disadvantages Restricted to proteins 104 Da MW Cannot detect prot
13、eins expressed at low levels Limited to 600800 separate spots Gel to gel reproducibility is poor Quantitation is poor, 50% or worse Dynamic range is limited, 10X Analysis is not directly coupled to separation,2-D PAGE AND MASS SPECTROMETRY IS NOT THE IDEAL TECHNIQUE FOR PROTEOMICS:,minutes,FIRST DIM
14、ENSION: SIZE-EXCLUSION CHROMATOGRAPHY,Fraction collected every 30 sec.,HYBRID 2-D SEPARATION: IEF-HPLC,Lecchi et. al. J.Biol Bioph. Meth. 2003,Sample: E. coli extractFirst dimension: y-axes IEF (Biorad rotofor) Second dimension: x-axes IEF fractions separated by HPLC (reversed phase C-18),to improve
15、 the efficiency of the proteolytic digestion it is important to reduce and alkylate disulfide bonds,s,s,s,s,s,s,s,s,s,s,s,s,s,s,s,R,R,R,R,R,R,reduction,alkylation,enzymatic digestion,R,R,s,Enzymatic digestion,*Some proteolytic enzymes are very specific, e.g.: trypsin cuts only at Lys-X or Arg-X Lys-
16、C Lys-X Arg-C Arg-X Glu-C (V-8) Glu-X and Asp-X,Peptide Mass Fingerprinting (PMF) by MALDI-TOF.,MS analysis of proteolytic fragments is a common way to identify a protein.,The following masses are entered for protein identification: 737.99 - 874.44 - 936.48 - 1030.65 - 1047.06 - 1153.67 - 1269.76 -
17、1428.85 1536.76 - 1676.97 - 1808.06 - 1862.99 - 2163.26 - 2274.28 Low MW peaks (e.g. 500 Da) are not generally used because of the high interference of the matrix.,the most common procedure to identify proteins by MS,Search Engine for protein identification: MASCOT (),Search Engine for protein ident
18、ification: ProFound (http:/prowl.rockefeller.edu),Mascot Search ResultsUser : paolo lecchi Email : phmpxlgwumc.edu Search title : test Database : MSDB 20040123 (1407720 sequences; 450709685 residues) Timestamp : 9 Apr 2004 at 02:40:25 GMT Top Score : 66 for 2HFLY, lysozyme (EC 3.2.1.17) (with IgG1 F
19、ab fragment hyHEL-5), chain Y - chickenProbability Based Mowse Score Ions score is -10*Log(P), where P is the probability that the observed match is a random event. Protein scores greater than 74 are significant (p0.05).Concise Protein Summary ReportHelpSignificance threshold p Max. number of hits2H
20、FLY Mass: 14305 Score: 66 Expect: 0.4 Queries matched: 5 lysozyme (EC 3.2.1.17) - chicken 1AT51 Mass: 10989 Score: 53 Expect: 6.7 Queries matched: 4 lysozyme (EC 3.2.1.17) chicken LZCH Mass: 16228 Score: 50 Expect: 13 Queries matched: 4 lysozyme (EC 3.2.1.17) c precursor chicken 1MELL Mass: 14035 Sc
21、ore: 48 Expect: 21 Queries matched: 4 lysozyme (EC 3.2.1.17), chain L - chickenSearch Parameters Type of search : Peptide Mass Fingerprint Enzyme : Trypsin Mass values : Monoisotopic Protein Mass : Unrestricted Peptide Mass Tolerance : 0.4 Da Peptide Charge State : 1+ Max Missed Cleavages : 1 Number
22、 of queries : 12,Protein ViewMatch to: 2HFLY Score: 70 Expect: 0.15 lysozyme (EC 3.2.1.17) (with IgG1 Fab fragment hyHEL-5), chain Y - chickenNominal mass (Mr): 14305; Calculated pI value: 9.18 NCBI BLAST search of 2HFLY against nr Unformatted sequence string for pasting into other applicationsTaxon
23、omy: Gallus gallusCleavage by Trypsin: cuts C-term side of KR unless next residue is P Number of mass values searched: 14 Number of mass values matched: 5 Sequence Coverage: 32%Matched peptides shown in Bold Red1 KVFGRCELAA AMKRHGLDNY RGYSLGNWVC AAKFESNFNT QATNRNTDGS 51 TDYGILQINS RWWCNDGRTP GSRNLCN
24、IPC SALLSSDITA SVNCAKKIVS 101 DGDGMNAWVA WRNRCKGTDV QAWIRGCRLStart - End Observed Mr(expt) Mr(calc) Delta Miss Sequence14 - 21 1030.65 1029.64 1029.51 0.13 1 RHGLDNYR 15 - 21 874.44 873.43 873.41 0.02 0 HGLDNYR 34 - 45 1428.85 1427.84 1427.64 0.20 0 FESNFNTQATNR 62 - 68 936.48 935.47 935.37 0.10 0 W
25、WCNDGR 98 - 112 1676.97 1675.96 1675.78 0.19 0 IVSDGDGMNAWVAWR No match to: 737.99, 1047.06, 1153.67, 1479.66, 1536.76, 1808.06, 1826.29, 2163.26, 2274.28,MALDI Post Source Decay (PSD) analysisstructural information (MS/MS) by MALDI,The fragmentation of a selected ion is a way to improve the confide
26、nce in identifying a protein by MS, or to get structural information. In this example an ion with m/z 1535 was selected and analyzed in PSD mode All the ions detected are obtained from the fragmentation of the selected m/z 1535 “parent ion”.,All the ions produced from the ion source have the same ki
27、netic energy (Ke) Kinetic energy = z(acVolt) = 1/2 mv2 z= ion charge velocity = s/t s= length of the flight path (e.g. 1m) measuring the time (t) an ion takes to travel through the flight tube (“field free region”) gives an accurate measurement of its mass (m).,Time-of-Flight (TOF) mass spectrometry
28、,The mass spec resolution can be increased by increasing the length of the flight tube or (better) by refocusing the ion beam with a “reflectron” (Mamryns mirror),Reflectron,detector,.an ion with high internal energy may fell apart after leaving the ionic source (“metastable ion”). The fragments pro
29、duced have all the same velocity. Therefore, in liner mode, they reach the detector at the same time. With a reflectron these fragment-ions are separated and detected at different time. The spectrum obtained provides structural information (post source decay analysis),The use of a a “reflectron” all
30、ows to obtain structural information,MALDI and Post Source Decay (PSD),Liquid Chromatography interfaced to Mass spectrometry (LC-MS),LC-MS/MS (DATA DEPENDENT ANALYSIS),SUMMARY2HTML v.8 (rev. 1.3), Copyright 1996-2000 Molecular Biotechnology, Univ. of Washington, J.Eng/J.Yates Licensed to Finnigan Co
31、rp., A Division of ThermoQuest Corp. 04/07/04, 02:49 PM, D:SEQUESTdog.fasta, AVG/AVGDatabase type = Peptide Start_scan=662; End_scan=676; Precursor_mass_tol =1.40; Group_scan_tol =1; Min_group_count =2; Min_ion_count =35; Low_mass_limit =300.00; High_mass_limit =4000.00; peptide_tol=1.0000; Fragment
32、_Ion_Tol=3.0000; B_ion=Yes; Y_ion=Yes; Enzyme:None selected# File* MassA* Xcorr* DelCn* Sp 1 HemDogSam5DD.0665.0671.1 1127.3 (-0.7) 1.3726 0.724 846.9RSp Ions Reference* MW* Sequence*1 13/ 18 gi|122579|sp|P02056|HBB_CANFA 15978 -.VHLTAEEKSL.V 1. gi|122368|sp|P01952|HBA_CANFA 20.0 (0,1,2,0,0) : 1 : 1
33、 1 2. gi|122579|sp|P02056|HBB_CANFA 17.0 (1,0,1,0,0) 1 : : 1 6.0 (0,0,1,0,0) : : 1 ,Search Engine for MS/MS: Syquest,MASS SPECTROMETRY IS AN ESSENTIAL TOOL IN PROTEIN ANALYSIS (AND PROTEOMICS),In particular two IONIZATION TECHNIQUES: MALDI (Matrix Assisted Laser Desorption Ionization) ESI (Electro Spray Ionization),SAMPLE PREPARATION IS VERY IMPORTANT (“GIGO”),BIOINFORMATIC TOOLS ARE FUNDAMENTAL FOR DATA PROCESSING.,CONCLUSIONS:,Paolo Lecchi phmpxlgwumc.edu 202-994 2933,
