Edman Sample Preparation Part 2. Bill Henzel Genentech Inc.
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1 Edman Sample Preparation Part 2 Bill Henzel Genentech Inc.
2 Determining a Protein is Blocked Estimate the amount of protein applied to the protein sequencer. Compare the staining intensity of the band of interest to a standard. Does the amino acid background increase with time? Is the protein sequencer working? Can other bands on the electroblott with similar staining intensity be sequenced?
3 Common Blocking Groups Pyroglutamic Acid Acetyl Ser, Ala, Met, Thr, Gly
4 Identification of Blocked Proteins Pyroglutamte aminopeptidase deblocking (pyrococcus furiosus) 1hr at 90 C Rapid enzymatic cleavage (5-30 min.) and Edman degradation. Chemical cleavage methods: CNBr Met/X Dilute Acid Asp/Pro Hydroxylamine Asn/Gly Iodosobenzoic acid,dmso/hcl Trp/X
5 Deblocking Protein Containing N-Terminal Pyroglutamic Acid Block PVDF membrane with 1% PVP-360 at 25 C on a shaker for 20 Minutes. Cleave with 1 mu of Pyrococcus furiosus pyroglutamate aminopeptidase in 50 mm sodium phosphate at 90 C for 1 hr.
6 Sequence Sorting of A Simple Protein Mixture from a Single Band
7 Sequence Sorting of a Complex Mixture
8 CNBr Cleavage of Blocked Proteins Wet PVDF with 1µl of methanol. Add µl of 0.1 N HCl 45 C 3 hr. Speed-Vac to completely dry PVDF membrane. Journal of Biochem. Biophys. Methods (1980) 3,
9 Rapid Method for Generating Internal Sequence of N-terminal Blocked Proteins Asp-Pro Cleavage 10% acetic acid, 7M guanidine HCl ph C 1 hr +OPA (Blocking all AA s except Pro) Anal. Chem. 72, (2000)
10 Average Number of Asp-Pro sites per Protein Molecular Weight 20,000 30,000 40,000 50,000 60,000 70,000 80,000 90, , , , ,000 A Average Number of Methionine Residues per Protein Comparison of Asp/Pro and Met Cleavage Sites ,000 B 30,000 40,000 50,000 Molecular Weight 60,000 70,000 80,000 90, , , , ,000
11 Percentage of Proteins Containing Asp/Pro Sites 20,000 30,000 40,000 50,000 60,000 70,000 80,000 90, ,000 Percentage of Proteins Containing ASP-Pro Site(s) 150, , , Molecular Weight
12 Sequence Analysis of a Asp/Pro Cleavage Reaction of Human Acetylglucosaminyltr Poly-His HumanAcetylglucosam inyltrpoly-his
13 N-terminally Blocked Proteins Identified by Database Searching from Asp-Pro Cleavage Sequence Data Accession # RIP_HUMAN a Protein Name a Swissprot Database b Genebank Translated Database Serine-threonine protein kinase rip human Molecular weight (Da) No. sequences found/expected Starting Residue No. SY16_HUMAN a SY_16 Human small inducible cytokine a16 precursor LITB_HUMAN a lithostathine 1-beta precursor human 16,414 2/ NM_001330_1 a Cardiotrophin 1 human 21,227 2/ CAH2_BOVIN a Bovine carbonic anhydrase 28,508 2/ JW0047 b RAGE_MOUSE a HGF_HUMAN a AF_156884_1 a Class I cytokinase receptor precusor human Advanced glycosylation end product specific receptor precursor - mouse Hepatocyte growth factor - pre cursor human, alpha chain Receptor interacting - serine threonine kinase 3 human 57,157 3/ ,901 3/ AF265242_1 a Type-1 T cell cytokine receptor human 69,460 2/ ,959 3/ AF316597_1 a Paracaspase human 92,272 4/ PHS2_RABIT a Glycogen phosphorylase rabbit 97,158 3/ Sequence Found 11,184 1/1 91 DPNLPLLPTRN 40,431 2/ DPETWVDADLYCQN DPKKNRRWHWSSGS DPQTDSSVSLL DPFGLPSFSPP DPALXPLALVYG DPGSLLPNVLDY DPLEATVHWAP DPLEXLNWVRL 40,448 1/1 128 DPASELTASVPN DPALKIKTK DPHTRWEYC DPNIRVGYC DPKPALVTK DPELHVKLA DPEAWSRPQ DPLEATVHWAPP DPLEKLNWVRLP DPFAQQRPYEN DPGTAGPRVWY DPIRENLGKHW DPLQALPPSAAPT DPIQGTEYSAESL DPKDANKGTPEET DPFHGVYHSHPGN DPXRIYYLSLEF DPVRTNFDAFPD DPVVGDRLRVIF
14 Rapid Enzymatic Cleavage of Blocked Proteins Wet PVDF with 1-2 µl of Methanol. Block with 0.5 ml of 1% PVP-360. Add 0.2 µg of Trypsin, Lys-N, Asp-N, Glu-C, Lys-C or Arg-C. Incubate 5-20 minutes at 37 C. Remove buffer and wash with water. Sequence peptides mixtures remaining on PVDF membrane.
15 Protein Identification using Rapid Enzymatic Cleavage Lys-N Digestion 37 C 10 min 1 KPSASNAFMV 0.83 pmol 2 KLYVYNDGYL 0.55 pmol P_AAE03653<P_AAE03653 Hum an extrace lularm atrix and ce ladhesion m olecule-17 <510 residues M R PG LSFLLALLFFLG Q AAG D LG D VG PPIPSPGFSSFPGVDSSSSFSSSSRSGSSSSRSLGSGGSVSQLFSN FTGSVDDR GTCQCSVSLPDTTFPVDRVERLEFTAHVLSQKFEKELSKVREYVQLISVYEKKLLNLTVRIDIMEKDTISYTELD FELIK VEVKEM EKLVIQLKESFGGSSEIVDQLEVEIRNMTLLVEKLETLDKNNVLAIRREIVALKTKLKECEASKDQNTP VVHPP PTPGSCGHGGVVNISKPSVVQLNW RGFSYLYGAW GRDYSPQHPNKGLYW VAPLNTDGRLLEYYRLYNTLD D LLLYIN AR E LR ITYGQGSGTAVYNNNMYVNMYNTGNIARVNLTTNTIAVTQTLPNAAYNNRFSYANVAW QDIDFAVDENGL WVIYSTEA STGNM VISKLNDTTLQ VLNTW YTKQ YKPSASNAFM VCGVLYATRTMNTRTEEIFYYYDTNTGKEGKLDIVM H KMQEKVQS
16 Summary of Protein Identification Methods Unknown Protein or Mixture of Proteins Reduction/Alkylation in Solution SDS-PAGE Electroblot LC_MS/MS SEQUEST FRAGFIT MASSPEP Edman Degradation Enzymatic Digestion Zip-Tip Sequence Found BLAST MOTIF Sequence Fractions BLAST MOTIF PSD FRAGFIT MASSPEP Peptide Collection MALDI-MS FRAGFIT Identification Mass Dependent MS/MS SEQUEST FRAGFIT MASSPEP
17 Acknowledgements Victoria Pham Wendy Sandoval Suzy Wong
18 Protein identification based on similarity to amino acid composition Each amino acid has an assigned weight, which were derived from a statistical analysis of the difference of the theoretical and experimental mole percentage values of 25 proteins. The molecular weight is also used as a weighting factor. A number of websites have similar programs some of which are listed on the ABRF website.
19 AminoSearch Search Results Protein Name Rank of match xtgfb.glhi.v5l.rk.theo 1 FGF19 1 HGH 1 HLFA1 1 Indian Hegehog 1 Myoglobin 1 MAMG 1 Complement Hlog 1 Chemokine beta 8 1 FKBP 1 VEGF 165 KDR 3
20 Distribution of Amino Acids in the N-terminal Region of Human Secreted Proteins % Change from Average Composition Cysteine Glutamine 1.8% 3.9% Proline Glutamic Acid 5.0% 6.1% Position Position
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