Laboration 1. Identifiering av proteiner med Mass Spektrometri. Klinisk Kemisk Diagnostik

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1 Laboration 1 Identifiering av proteiner med Mass Spektrometri Klinisk Kemisk Diagnostik Sven Kjellström 2014 kjellstrom.sven@gmail.com

2 Laboration 1 Klinisk Kemisk Diagnostik Identifiering av proteiner med peptide map fingerprint. Profilering av proteiner. Mötesrum Preparation av prover MS lab SAFETY: Labcoat and safety glasses Sven Kjellström 2014

3 Praktiskt arbete SAFETY: Labcoat and safety glasses Mix sample and matrix solution 0.5 ul ul on target. Collect MS and transfer peaklist to USB stick. Perform database search using mascot ( 2 ul 2 ul 2 ul 18 ul 18 ul Sven Kjellström ul 18 ul

4 Ionization of biomolecules ESI Electrospray ionization MALDI Matrix assisted laser Desorption/ionization Sample in liquid Purification is needed Sample in solid state Purification optinal Fenn et al. Science 1989, 246, Karas & Hillenkamp Anal. Chem ,

5 Identification of protein patterns in clinical chemistry

6 Identification of proteins in clinical chemistry MALDI is faster and more accurate than previous used methods. This saves time and money by making faster and safer determinations of bacteria infections.

7 What is a protein? Linear polymers built of 20 different aminoacids Defined conformation Protein: IgG Hemoglobin Insulin Weight kda: Amino acids: >

8 More difficult to measure large things MS is a sophisticated balance for many analytes including proteins +/ Da +/- 1 Da +/- 2 Da 1kDa 20kDa 60kDa accuracy MW Better sensitivity and accuracy More sample is is needed

9 Make peptides of proteins 8 M UREA Native protein

10 Use of enzyme gives peptides

11 Protein identification using peptide map fingerprint MS analysis determines the weight of individual peptides MS

12 Protein identification using PMF Experimental determined Theoretical determined MS MS Database search Peptide mass fingerprinting = PMF Peter James 1993

13 % Intensity % Intensity % Intensity Peptide map fingerprint PMF Reflector Spec #1[BP = , 76095] 7.6E+4 Simple and powerfull analysis method Three proteins different fingerprints Mass (m/z) 4700 Reflector Spec #1[BP = , 9241] E Mass (m/z) 4700 Reflector Spec #1[BP = , 60397] Protein identity can be obtained from clean samples. Some modifications can be detected E Pattern is recognized not the single peptides Mass (m/z) Sven Kjellström 2014 The protease trypsin cleaves peptides bonds specifically on the C-terminal side of Arginine (R) and Lysine (K)

14 Are all suggested peptides true in PMF? 1 MKTTVTKLLA TVAAASTIFG MSTLPAFAAE GKSASNGNSV NISDVNATAE 51 TRALFDKLKN SGKGDLRFGQ QHATDENISS SASQGDVYET TGKYPAVFGW 101 DAGLALRGAE KPGSGADKNA NAKALAQNIT DADSKGAIVT LSAHWCNPGT 151 GKDFNDTTAV ASELLPGGKY SGTFNKELDA IAATAQRAKR SDGTLIPIIF 201 RPLHENNGSW FWWGATHASA SEYKELYRYI VDYLRDVKDV HNLLYAYSPG 251 GVFNGDSTDY LATYPGDQWV DVLGYDEYDS DDSADDSSAW INTVVKDMKM 301 VSDQASQRGK IVALTEFGRS GDRKFKESGT GDKDTKFFSE LAEALAENVP 351 STAYMMTWAN FGGGGDNFQA YTSWKGSDGE ADFKAFADSN KNLMASKDNV 401 DYSNAPAAAM QNGSARIVTP VDGNRVTDTK VVVRVKTEGV KYSDLDLNSA 451 IVTTDRGQNV KLKYSCNGYF TGILDLNAAG INLDQSKLTL TPQVKTKDGK 501 TLAAADGNGS VTVKLGAKPE QTVDNVEDFD SYDNEAELQS VYSPSHSTKS 551 NLTLVDSPED NGTKAGNIHY DFVSYPEYNG FQRSHTPKQD WSGFSKLNMF 601 LKADGSDHKF VVQVNAGGVT FEAYPKIDGT DGHVVSLNFG DADGNGGDFA 651 PASWDTAHAG MKLSQKLLSK VGSFALYIND NGGNRPKSGD LTLDSIKLDG 701 KRDAYAPNTN PTPGNTAKAQ SVDDFSGYSD DAAAQSAWGN RGHTEVLSLD 751 EGPTDGSKAL RFKYDFSNGG WYDVAKYLDG ANWSGESVLA FQVKGDGSGN 801 AIGLQIGTSD GKYFLASVKL DFTGWKQIEI PLVDNANLTQ SWPEDANKDN 851 PMTEDDLASI KELVFASQQW NSESDGLDSS IADIKVEPAE NTSNEQTPKD 901 ESKTEVKADK EQEQSEDTSA DVTAQDPATC PISDEDSKGS TGNTTVTVKP 951 TPDTKEPADN TGKDGLSRTG SNIISAIAAV AVLLLGGCAV LIARKRKGGD 1001 IE SEQ Meassuerd ppm, miss cleavege Seq K.SASNGNSVNISDVNATAETR.A R.FGQQHATDENISSSASQGDVYETTGK.Y K.DFNDTTAVASELLPGGK.Y K.YSGTFNK.E K.ELDAIAATAQR.A R.YIVDYLR.D K.MVSDQASQR.G K.IVALTEFGR.S K.ESGTGDKDTK.F K.DNVDYSNAPAAAMQNGSAR.I R.IVTPVDGNR.V K.YSDLDLNSAIVTTDR.G R.FKYDFSNGGWYDVAK.Y Pattern is recognized not the single peptides Result from database search. Sven Kjellström 2014 Number of mass values searched: 36 Number of mass values matched: 13 Sequence Coverage: 17% Matched peptides shown in Bold Red

15 % Intensity % Intensity Several peaks are artefacts in PMF Missed cleavage R.FKYDFSNGGWYDVAK.Y Largest mass deviation Not a peptide in MS spectra isotopic pattern 54.4 Peptide isotopic pattern around Reflector Spec #1[BP = , 60397] Low signal intensity E Mass (m/z) Sven Kjellström 2014 Number of mass values searched: 36 Number of mass values matched: 13 Sequence Coverage: 17% 12 In conclusion we don t believe in this peptide. MSMS can confirm if this is the right sequence.

16 % Intensity Check for Methionine oxidation DMQLGR 16 Da mass shift DMQLGR methionie oxidation 5.8E Mass (m/z) Sven Kjellström 2014

17 Modification of proteins are detectable M- M- M- M- Ox- Metionine Native Oxidation MS MS + 16 Da Native Oxidation

18 Sven Kjellström 2014 Precision & Accuracy