Laboration 1 Identifiering av proteiner med Mass Spektrometri Klinisk Kemisk Diagnostik Sven Kjellström 2014 kjellstrom.sven@gmail.com 0702-935060
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
Praktiskt arbete SAFETY: Labcoat and safety glasses Mix sample and matrix solution 0.5 ul + 0.5 ul on target. Collect MS and transfer peaklist to USB stick. Perform database search using mascot (http://www.matrixscience.com/search_form_select.html). 2 ul 2 ul 2 ul 18 ul 18 ul Sven Kjellström 2014 18 ul 18 ul
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, 64-71 Karas & Hillenkamp Anal. Chem. 1988 60, 2299-2301.
Identification of protein patterns in clinical chemistry
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. Bo.Nilson@med.lu.se
What is a protein? Linear polymers built of 20 different aminoacids Defined conformation Protein: IgG Hemoglobin Insulin Weight kda: 150 58 5.8 Amino acids: >1000 574 51 7
More difficult to measure large things MS is a sophisticated balance for many analytes including proteins +/- 0.02 Da +/- 1 Da +/- 2 Da 1kDa 20kDa 60kDa accuracy MW Better sensitivity and accuracy More sample is is needed
Make peptides of proteins 8 M UREA Native protein
Use of enzyme gives peptides
Protein identification using peptide map fingerprint MS analysis determines the weight of individual peptides MS
Protein identification using PMF Experimental determined Theoretical determined MS MS Database search Peptide mass fingerprinting = PMF Peter James 1993
1013.4199 1032.4849 1045.4790 1090.4866 1098.4973 1118.5057 1137.4548 1159.5410 1193.4332 1195.5703 1221.5771 1246.5221 1251.5968 1272.6118 1283.6082 1303.5791 1320.5492 1351.6531 1367.5649 1412.6182 1439.6052 1456.6154 1479.6976 1494.7115 1506.6615 1523.6591 1566.3805 1580.6945 1594.5793 1608.7203 1633.7640 1641.7312 1660.6541 1708.7596 1736.8038 1753.7898 1775.7819 1814.7139 1832.7039 1857.6085 1871.8499 1897.7012 1927.5537 1951.8038 1958.8528 1979.9430 1337.5684 % Intensity 1384.6035 1759.8145 1267.5990 1011.2650 1022.2769 1050.2594 1116.3801 1141.2794 1163.4685 1185.4189 1249.4391 1283.5400 1288.1722 1305.5375 1327.4531 1347.3284 1367.4131 1386.4127 1399.3271 1423.3110 1443.2267 1461.6013 1493.5682 1511.6558 1537.6064 1549.5404 1569.1527 1580.5801 1597.5599 1646.2312 1667.6017 1703.6183 1723.6069 1789.6676 1851.6627 1873.4775 1823.6670 1888.7302 1940.5563 1964.7122 1639.7318 % Intensity 1567.5428 1439.6255 1479.6060 1040.8270 1067.4137 1092.4552 1105.3752 1125.3170 1136.4276 1152.1351 1172.7208 1189.3888 1230.3871 1280.2285 1301.5452 1313.4772 1339.4727 1350.5405 1433.5923 1456.4698 1495.4891 1561.6816 1584.5533 1616.1597 1631.4069 1649.4016 1698.5775 1712.6016 1826.6677 1840.6794 1598.5629 1734.8011 1470.4771 1478.6273 1606.7125 % Intensity 1296.5441 1633.4099 1168.4790 Peptide map fingerprint PMF 100 90 80 70 4700 Reflector Spec #1[BP = 1168.5, 76095] 7.6E+4 Simple and powerfull analysis method. 60 50 40 30 20 Three proteins different fingerprints 10 0 1000 1200 1400 1600 1800 2000 Mass (m/z) 4700 Reflector Spec #1[BP = 1479.6, 9241] 100 9.2E+3 90 80 70 60 50 40 30 20 10 0 1000 1200 1400 1600 1800 2000 Mass (m/z) 4700 Reflector Spec #1[BP = 1267.6, 60397] Protein identity can be obtained from clean samples. Some modifications can be detected. 100 6.0E+4 90 80 70 60 50 40 30 20 10 Pattern is recognized not the single peptides. 0 1000 1200 1400 1600 1800 2000 Mass (m/z) Sven Kjellström 2014 The protease trypsin cleaves peptides bonds specifically on the C-terminal side of Arginine (R) and Lysine (K)
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 33-52 2006.9467 2005.9394 2005.9301 5 0 K.SASNGNSVNISDVNATAETR.A 68-93 2757.2444 2756.2371 2756.2162 8 0 R.FGQQHATDENISSSASQGDVYETTGK.Y 153-169 1734.8612 1733.8539 1733.8472 4 0 K.DFNDTTAVASELLPGGK.Y 170-176 816.3779 815.3707 815.3814-13 0 K.YSGTFNK.E 177-187 1158.6167 1157.6094 1157.6040 5 0 K.ELDAIAATAQR.A 229-235 941.5090 940.5018 940.5018-0 0 R.YIVDYLR.D 300-308 1021.4761 1020.4688 1020.4658 3 0 K.MVSDQASQR.G 311-319 1005.5718 1004.5646 1004.5655-1 0 K.IVALTEFGR.S 327-336 1037.4781 1036.4709 1036.4673 3 1 K.ESGTGDKDTK.F 398-416 1951.8679 1950.8606 1950.8490 6 0 K.DNVDYSNAPAAAMQNGSAR.I 417-425 970.5259 969.5187 969.5244-6 0 R.IVTPVDGNR.V 442-456 1682.8329 1681.8256 1681.8159 6 0 K.YSDLDLNSAIVTTDR.G 762-776 1796.7859 1795.7786 1795.8206-23 1 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
1752.7739 1753.7898 1758.8158 % Intensity % Intensity 1759.8145 1796.7859 Several peaks are artefacts in PMF Missed cleavage 762-776 1796.7859-23 1 R.FKYDFSNGGWYDVAK.Y 100 90 80 Largest mass deviation Not a peptide in MS spectra isotopic pattern 54.4 Peptide isotopic pattern around 1700 4700 Reflector Spec #1[BP = 1267.6, 60397] 70 60 50 Low signal intensity 100 90 80 70 60 4.5E+4 50 40 40 30 30 20 10 20 0 1751.0 1755.8 1760.6 1765.4 1770.2 1775.0 Mass (m/z) 10 0 1781.0 1790.4 1799.8 1809.2 1818.6 1828.0 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.
673.271 781.264 1055.505 1365.642 1526.803 1949.909 904.468 % Intensity 735.337 719.364 Check for Methionine oxidation 100 90 80 70 69-74 719.343 DMQLGR 16 Da mass shift 69-74 735.343 DMQLGR methionie oxidation 5.8E 60 50 40 30 20 10 0 599.0 919.2 1239.4 Mass (m/z) 1559.6 1879.8 2200.0 Sven Kjellström 2014
Modification of proteins are detectable M- M- M- M- Ox- Metionine Native Oxidation MS MS + 16 Da Native Oxidation
Sven Kjellström 2014 Precision & Accuracy