TÁMOP-4.1.1.C-13/1/KONV-2014-0001 projekt Az élettudományi-klinikai felsőoktatás gyakorlatorientált és hallgatóbarát korszerűsítése a vidéki képzőhelyek nemzetközi versenyképességének erősítésére program keretében finanszírozott LABORATÓRIUMI GYAKORLAT SILLABUSZ SYLLABUS OF A PRACTICAL DEMONSTRATION financed by the program Practice-oriented, student-friendly modernization of the biomedical education for strengthening the international competitiveness of the rural Hungarian universities Dátum / Date: Helyszín / Place: 2018. OKTÓBER 03. / OCTOBER 03, 2018 SZBK KÖZPONTI LABORATÓRIUMOK, 031. LAB. / LAB # 031, CORE FACILITIES, BRC SZEGED, TEMESVÁRI KRT. 62. Gyakorlati foglalkozás címe / Title of the practical demonstration: MASS SPECTROMETRY BASED PROTEOMICS (MASS SPECTROMETRY BASED PROTEIN IDENTIFICATION PRACTICAL) Gyakorlatvezető / Demonstrator: ÉVA HUNYADI-GULYÁS Biological Research Centre Address: H-6726 Szeged, Temesvári krt. 62. Mail: H-6701 Szeged, POB 521. www.brc.hu
Aim of this practical course: Identify the protein content of Sample #1 and Sample #2 Sample #1: Tryptic digest of a bovine protein. Before digestion the protein was reduced with dithiotreitol (DTT) and free sulfhydryls were alkylated with Iodoacetamide (IAM). (it means that all the cysteine residues are carbamidomethylated finally (+57 Da)) Sample #2: Tryptic digest of a protein mix from different species. Before digestion the protein mix was reduced with DTT (dithiotreitol) and free sulfhydryls were alkylated with Iodoacetic acid (IAA). (it means that all the cysteine residues are carboxymethylated (+58 Da)) During the practical we use mass spectrometry based protein identification. Both of the samples were digested before. 1. MALDI-TOF (Matrix Assisted Laser Desorption/Ionisation Time Of Flight) measurement will be performed for peptide-mass-fingerprint type analysis. 2. LC-MSMS ( liquid chromatography coupled tandem mass spectrometry) experiment will be demonstrated, data evaluation will be performed. Useful websites: http://www.ms-textbook.com/ http://www.uniprot.org/ http://expasy.org/ http://ionsource.com/ Literature: Michael Kinter and Nicholas E. Sherman: Protein Sequencing and Identification Using Tandem Mass Spectrometry. Wiley-Interscience, 2000. ISBN: 978-0-471-32249-8 page 2
Module #1 : Spot samples to the MALDI target plate Chemicals: (all solvent should be as pure as possible) Acetonitrile (HPLC grade) (ACN) Water (HPLC grade) Trifluoro-acetic acid (TFA) 2,5-Dihydroxybenzoic acid (DHB) Matrix solution: 20 mg/ml DHB in 10% ACN Standard peptide mix Unknown samples #1 and #2 Spotting procedure: Spot 0.75 μl of matrix solution to a position of the target plate and spot on the top of that 0.75 μl of sample. Mix together with the help of the pipet (aspirate and dispense 3 times). Let it dry at room temperature. Spot the 2 unknown samples and the peptide standard mix onto the target plate to three different positions (the standard should be in the middle of the two unknown samples), according to the procedure. Module #2 MALDI-TOF analysis calibration Instrument: Bruker Reflex-III MALDI- TOF mass spectrometer 1. Acquire a mass spectrum from the standard peptide mix using the RP 2-3kDa_med parameter set. Calibrate the instrument. Standard peptide mix contains the following peptides: Peptide [M+H]+ Mono isotopic [M+H]+ Average Angiotensin II 1046.5418 1047.19 Angiotensin I 1296.6848 1297.49 Substance P 1347.7354 1348.64 Bombesin 1619.8223 1620.86 ACTH clip 1-17 2093.0862 2094.43 ACTH clip 18-39 2465.1983 2466.68 Somatostatin 28 3147.4710 3149.57 page 3
2.0 1.5 1.0 0.5 0.0 1000 1250 1500 1750 2000 2250 2500 2750 3000 3250 Module #3 MALDI-TOF analysis of the unknown samples Intens. [a.u.] x10 4 847.598 1001.642 1089.570 1164.581 1286.700 1407.756 1639.951 2060.170 2147.157 1479.834 1490.850 2438.417 2390.315 1751.025 1543.883 1905.053 2521.401 2727.237 MALDI-TOF spectrum m/z Instrument: Bruker Reflex-III MALDI- TOF mass spectrometer 1.Using the same parameter set acquire spectra from the unkown samples. 2. Evaluate the mass spectra using manual peak picking. (Label the monoisotipic peaks.) 3. Print the spectra. Module #4 Peptide mass fingerprint database search Using ProteinProspector s MSFit module (http://prospector.ucsf.edu/prospector/mshome. htm ) or MatrixScience MASCOT Peptide Mass Fingerprint module (http://www.matrixscience.com ) start a database search using the Swissprot database. Note the following parameters: enzyme, mass error, modifications, instrumentation! Database search engines Results Sample #1: Sample #2: page 4
140812_43_6PM #2543 RT: 16.90 AV: 1 NL: 1.96E3 T: ITMS + c NSI d w Full ms2 797.87@cid35.00 [205.00-1610.00] 724.3961 1900 1800 1700 1600 1087.5244 1500 837.4473 1400 1300 1200 552.2567 1100 1365.7045 1000 437.1816 900 800 1000.5093 1467.6724 700 1202.6024 600 508.2943 1158.5193 871.4973 500 1043.4606 667.3083 758.3557 400 300 899.6758 200 380.1329 940.5127 100 237.0626 357.2037 577.3461 1321.3895 1572.6345 0 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 m/z Module #5 - Demonstration of the LC-MSMS analysis of Sample #2 Instrument: Waters NanoAcquity HPLC coupled to a LTQ-Orbitrap-Elite mass spectrometer Notes / instructions continued: Module #6 - Manual analysis of an MSMS spectrum Intensity CID spectrum of the peak at m/z 797.8735, z=2+ Instrument: Waters NanoAcquity HPLC coupled to a LTQ-Orbitrap-Elite mass spectrometer Notes / instructions continued: page 5