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Phosphoprotein Analysis Standardized Approach for Identification of Focal Adhesion Proteins’ Phosphorylation Sites Antonio F. M. Pinto, David M. Smalley, Li Ma, Pablo R. Grigera, Nicholas E. Sherman, J. Thomas Parsons and Jay W. Fox. University of Virginia, Charlottesville, VA. 6112 Table II. Phosphorylation site coverage of seven proteins analyzed over the past year by the Protein C18 4.0E+07 Discovery Initiative. Cell Migration Consortium data are compared with two phosphorylation 4684 databases, PhosphoSITE (http://www.phosphosite.org) and Phospho.ELM 3.5E+07 4716 (http://phospho.elm.eu.org). Total, unique and shared number of phosphorylation sites are indicated. 3.0E+07 4744 5095 y 5968 Abstract t Protein Discovery Initiative PhosphoSITE Phospho.ELM i 2.5E+07 5120 s Protein n e total(unique/shared) total(unique/shared) total(unique/shared) t 4508 5141 n I 2.0E+07 β−PIX 16(13/3) 6(3/3) 2(1/1) 1.5E+07 5869 CORTACTIN 18(12/6) 25(19/6) 8(7/1) The Protein Discovery Initiative goal is to develop a census of the migration proteome by FAK 25(19/6) 14(8/6) 11(7/4) 1.0E+07 4222 5492 identifying interactions and post-translational modifications, i.e. phosphorylation, of novel 3064 GIT1 32(25/7) 10(3/7) 4(1/3) 0.5E+07 3020 3184 3806 PAK 3(0/3) 11(8/3) 1(1/0) molecules involved in cell migration. Over the past few years, identification of these 2724 2869 3227 38 318 452 665 785 1141 1359 1523 1890 2202 2413 PAXILLIN 45(13/32) 51(19/32) 7(0/7) phosphorylation sites have been done by a variety of methodologies, such as mutation of 0 0 10 20 30 40 50 60 70 80 90 100 110 120 TALIN 31(26/5) 8(4/4) 1(0/1) Time (min) phosphorylation sites, phospho-antibodies, electrophoretic mobility shifts, immobilized metal gi|1722836|sp|P42768|WASP_HUMAN (100%), 54451.5 Da Wiskott-Aldrich syndrome protein (WASp) ion affinity chromatography (IMAC), titanium dioxide (TiO2) affinity chromatography and LC- 29 unique peptides, 107 unique spectra, 526 total spectra, 338/515 amino acids (66% coverage) Table III. Phosphorylation Sites in Paxillin. Phosphorylation sites identified by the Protein Discovery MS/MS. Over the past year, we have developed a standard approach for phosphopeptide D Y K D D D D K E G V R T M S G G P M G G R P G G R G A P A V Q Q N I P S T L L Q D H E N Q R L F E M L G R K C L T L A T A V V Q L Y L A L P P G A E H W T K E H C G A V C F V K D N P Q K S Y F I R L Initiative are compared to data from PhosphoSITE and Phospho.ELM databases. Phosphorylation sites Y G L Q A G R L L W E Q E L Y S Q L V Y S T P T P F F H T F A G D D C Q A G L N F A D E D E A Q A F enrichment using direct LC-MS/MS and TiO2 affinity chromatography coupled with LC- R A L V Q E K I Q K R N Q R Q S G D R R Q L P P P P T P A N E E R R G G L P P L P L H P G G D Q G G are indicated in red; sites present in more than one database are highlighted in yellow. P P V G P L S L G L A T V D I Q N P D I T S S R Y R G L P A P G P S P A D K K R S G K K K I S K A D MS/MS. I G A P S G F K H V S H V G W D P Q N G F D V N N L D P D L R S L F S R A G I S E A Q L T D A E T S K L I Y D F I E D Q G G L E A V R Q E M R R Q E P L P P P P P P S R G G N Q L P R P P I V G G N K G Reverse phase chromatography in C18 column was used to analyze a tryptic digest of R S G P L P P V P L G I A P P P P T P R G P P P P G R G G P P P P P P P A T G R S G P L P P P P P G A G G P P M P P P P P P P P P P P S S G N G P A P P P L P P A L V P A G G L A P G G G R G A L L D Q Wiskott-Aldrich Syndrome Protein (WASP). The phosphopeptide enrichment method I R Q G I Q L N K T P G A P E S S A L Q P P P Q S S E G L V G A L M H V M Q K R S R A I H S S D E G E D Q A G D E D E D D E W D D modified from Canti et al. (Anal. Chem., 2007, 79:4666-4673) with or without the addition of 3996 2,5-dihydroxybenzoic acid (DHB) was used to isolate and identify phosphorylation sites. 3898 2.2E+07 TiO enrichment 3861 Protein coverage and spectra counting of these three runs were used to compare the 2 3780 efficiency of the different methodologies. 2.0E+07 1.8E+07 Finally, we compared phosphorylation sites identified in the Protein Discovery Initiative 1.6E+07 with results found in databases (PhosphoSITE, http://www.phosphosite.org, and 1.4E+07 y t i s n 1.2E+07 Phospho.ELM, http://phospho.elm.eu.org). Thus far, using our established methodologies, e t n we have found a number of phosphorylation sites in the proteins studied which were not I 1.0E+07 0.8E+07 3755 reported in the other two databases. 2661 0.6E+07 3706 0.4E+07 2842 3686 2863 3546 2101 2515 0.2E+07 1778 2086 2219 1474 1499 29 178 308 400 505 678 783 886 1042 1219 0 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 Time (min) gi|1722836|sp|P42768|WASP_HUMAN (100%), 54451.5 Da Wiskott-Aldrich syndrome protein (WASp) TiO2 Methods 17 unique peptides, 54 unique spectra, 72 total spectra, 235/515 amino acids (46% coverage) D Y K D D D D K E G V R T M S G G P M G G R P G G R G A P A V Q Q N I P S T L L Q D H E N Q R L F E M L G R K C L T L A T A V V Q L Y L A L P P G A E H W T K E H C G A V C F V K D N P Q K S Y F I R L Y G L Q A G R L L W E Q E L Y S Q L V Y S T P T P F F H T F A G D D C Q A G L N F A D E D E A Q A F R A L V Q E K I Q K R N Q R Q S G D R R Q L P P P P T P A N E E R R G G L P P L P L H P G G D Q G G TiO2 Columns P P V G P L S L G L A T V D I Q N P D I T S S R Y R G L P A P G P S P A D K K R S G K K K I S K A D I G A P S G F K H V S H V G W D P Q N G F D V N N L D P D L R S L F S R A G I S E A Q L T D A E T S Capillary column (360x150) of about 15cm with Kasil frit in one extremity were used. K L I Y D F I E D Q G G L E A V R Q E M R R Q E P L P P P P P P S R G G N Q L P R P P I V G G N K G R S G P L P P V P L G I A P P P P T P R G P P P P G R G G P P P P P P P A T G R S G P L P P P P P G Column was filled with 2cm of Titansphere (GL Sciences Inc.) packing material in 100% A G G P P M P P P P P P P P P P P S S G N G P A P P P L P P A L V P A G G L A P G G G R G A L L D Q I R Q G I Q L N K T P G A P E S S A L Q P P P Q S S E G L V G A L M H V M Q K R S R A I H S S D E G acetonitrile.
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