LABORATÓRIUMI GYAKORLAT SILLABUSZ SYLLABUS OF A PRACTICAL DEMONSTRATION. financed by the program

Transcription

1 TÁMOP C-13/1/KONV 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: 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

2 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: Literature: Michael Kinter and Nicholas E. Sherman: Protein Sequencing and Identification Using Tandem Mass Spectrometry. Wiley-Interscience, ISBN: page 2

3 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 Angiotensin I Substance P Bombesin ACTH clip ACTH clip Somatostatin page 3

4 Module #3 MALDI-TOF analysis of the unknown samples Intens. [a.u.] x 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 ( htm ) or MatrixScience MASCOT Peptide Mass Fingerprint module ( ) 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

5 140812_43_6PM #2543 RT: AV: 1 NL: 1.96E3 T: ITMS + c NSI d w Full ms @cid35.00 [ ] 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 , z=2+ Instrument: Waters NanoAcquity HPLC coupled to a LTQ-Orbitrap-Elite mass spectrometer Notes / instructions continued: page 5