Lab 1 - Klinisk Kemisk Diagnos9k
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1 Lab 1 - Klinisk Kemisk Diagnos9k Mass Spectrometry Quan%fica%on & Protein iden%fica%on by pep%de mass fingerprint melinda.rezeli@bme.lth.se
2 Lab 1 - Klinisk Kemisk Diagnos9k MALDI mass spectrometry Quan%fica%on by MALDI MS Protein iden%fica%on by pep%de mass fingerprint (PMF) Mee%ng room Sample prepara%on MS lab SAFETY: Labcoat and safety glasses
3 Mass Spectrometry Mass spectrometry is an analy%cal technique used to measure the mass-to-charge ra-o of ions. This is achieved by ionizing the sample and separa-ng ions of differing masses and recording their rela%ve abundance by measuring intensi%es of ion flux. A typical mass spectrometer comprises three parts, an ion source, a mass analyzer, and a detector system:
4 Ioniza9on of biomolecules ESI - Electrospray ioniza%on MALDI - Matrix assisted laser desorp%on/ioniza%on Sample in liquid Purification is needed Sample in solid state Purification optional Fenn et al. Science 1989, 246, Karas & Hillenkamp Anal. Chem ,
5 MALDI-LTQ Orbitrap XL
6 Orbitrap FT mass analyzer w = oscilla%on frequency k = instrumental const. m/z =. what we want! ω = k m / z
7 Quan9fica9on Standard curve
8 Standard curve without IS Concentration! pmol/µl! Mean! (Height)! Stdev! (Height)! RSD! (%)! 2.5" 2267" 728" 32.1" 2.0" 2059" 732" 35.5" 1.5" 1067" 312" 29.3" 1.0" 354" 81" 22.7" 0.5" 122" 54" 44.5" RSD < 45 %
9 Quan9fica9on by using stable isotope labeled standards L Analyte H SIS MS pep%des with 13C and/or 15N modified amino acids (arginine or lysine) chemically iden%cal to the target pep%de but with mass difference (similar behavior to the target pep%de with regards to chromatographic separa%on, ioniza%on, and fragmenta%on) SIS: Stable Isotope Standard PAR: Peak Area Ra%o PAR = Light (Analyte) Peak Area Heavy (SIS) Peak Area Analyte concentra%on = PAR * SIS pep%de concentra%on
10 Standard curve with IS Concentra9on pmol/µl! Mean (H A /H IS )! Stdev (H A /H IS )! RSD (%)! 2.5" 0.549" 0.017" 3.1" 2.0" 0.453" 0.027" 6.1" 1.5" 0.338" 0.027" 7.9" 1.0" 0.231" 0.038" 16.6" 0.5" 0.142" " 0.3" RSD < 17 %
11 Task 1 Genera%on of a standard curve 1. Prepare a 2 fold dilu%on series of sample 1 (light pep%de) in 5 steps 2. Prepare matrix solu%on with SIS (heavy pep%de). 3. Add 1 ul sample + 1 ul matrix (with SIS) solu%on on a MALDI target plate (MTP). 4. Collect MS data and transfer peaklist to USB s%ck. 5. Create standard curve in Excel with normaliza%on to SIS pep%de light stock 2x 4x 8x 16x 32x 2 ul SIS stock 38 ul matrix solu%on
12 Protein iden9fica9on More difficult to measure large things MS is a sophis%cated balance for many analytes including proteins +/ Da +/- 1 Da +/- 2 Da accuracy 1kDa 20kDa 60kDa MW Beler sensi%vity and accuracy More sample is is needed
13 Protein diges%on with specific a enzyme 8 M UREA enzyme (trypsin) Na%ve protein reduc%on/ alkyla%on (op%onal) pep%des
14 Importance of reduc%on/alkyla%on Disulfide bridges 2230 Da Da No reduc%on results in long branched pep%des! 7434 Da
15 MALDI-MS Non-reduced 100 AV_ _01 4 (0.130) Cm (1:5) ; TOF LD+ 1.15e PSA Sigma digest H2 Pulsevolt:2574.0Laser: 5.0 M@LDI AV_ _02 1 (0.030) Cm (1:5) 100 Reduced % ; Apr-2008 TOF LD+ 9.12e m/z % m/z
16 MS analysis of the proteoly%c pep%des 8 M UREA enzyme (trypsin) Na%ve protein reduc%on/ alkyla%on (op%onal) MS pep%des MS analysis determines the weight of individual pep%des
17 Protein iden%fica%on using PMF 8 M UREA enzyme (trypsin) Na%ve protein Experimental determined Theore%cal Determined MS MS pep%des Database search Pep%de mass fingerprin%ng = PMF
18 Pep%de mass fingerprint 4700 Reflector Spec #1[BP = , 76095] E % Intensity Mass (m/z) PMF from 3 different proteins % Intensity E Reflector Spec #1[BP = , 9241] Mass (m/z) Reflector Spec #1[BP = , 60397] E % Intensity Simple and powerfull analysis method. Protein iden%ty can be obtained from clean samples. PaXern is recognized not the single pep9des! Mass (m/z)
19 Database search result (MASCOT) 1 IVGGWECEKH SQPWQVLVAS RGRAVCGGVL VHPQWVLTAA HCIRNKSVIL! 51 LGRHSLFHPE DTGQVFQVSH SFPHPLYDMS LLKNRFLRPG DDSSHDLMLL! 101 RLSEPAELTD AVKVMDLPTQ EPALGTTCYA SGWGSIEPEE FLTPKKLQCV! 151 DLHVISNDVC AQVHPQKVTK FMLCAGRWTG GKSTCSGDSG GPLVCNGVLQ! 201 GITSWGSEPC ALPERPSLYT KVVHYRKWIK DTIVANP 72% sequence coverage! Start-End Observed Mr(expt) Mr(calc) ppm Miss Sequence! IVGGWECEK.H! K.HSQPWQVLVASR.G! R.AVCGGVLVHPQWVLTAAHCIR.N! K.SVILLGR.H! R.HSLFHPEDTGQVFQVSHSFPHPLYDMSLLK.N! R.HSLFHPEDTGQVFQVSHSFPHPLYDMSLLK.N Oxidation (M)! R.FLRPGDDSSHDLMLLR.L! R.FLRPGDDSSHDLMLLR.L Oxidation (M)! R.LSEPAELTDAVK.V! K.VMDLPTQEPALGTTCYASGWGSIEPEEFLTPK.K Oxidation (M)! K.KLQCVDLHVISNDVCAQVHPQK.V! K.FMLCAGR.W! K.FMLCAGR.W Oxidation (M)! K.VVHYR.K Score: 94
20 Task 2 Iden%fica%on of unknown proteins by Pep%de Mass Fingerprint (PMF) 1. Select 3 different unknown protein samples (ready digests). 2. Add 1 ul sample + 1 ul matrix solu%on on a MALDI target plate (MTP). 3. Collect MS data and transfer peaklist to USB s%ck. 4. Perform database search using mascot PMF hlp:// FORMVER=2&SEARCH=PMF
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23 Task 1 (Pep%de quan%fica%on) 1. Dilu%on series (5 dil. steps) light stock 2x 4x 2. Sample with unknown concentra%on (1 sample) 8x 16x 32x 1. Matrix mixed with the heavy pep%de standard 2 ul SIS stock 38 ul matrix solu:on 2. Sample with unknown concentra%on (1 sample) Task 2 (Pep%de Mass Fingerprint) 1. Unknown protein digests (3 samples) 1. Unknown protein digests (3 samples)
24 1a. Calibra%on curve genera%on 1b. Calcula%on of the unknown concentra%on of the samples (1 sample/group) 2. Database search, iden%fica%on of the unknown proteins (3 samples/group)
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