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. Columns were rinsed with water prior to use or storage. E D Q A G D E D E D D E W D D All column washing and loading was done at 500 psi with a flow rate of approximately 1-2 µL/mL. 0.9E+07 TiO2 + DHB enrichment
0.8E+07 TiO2 enrichment method (modified from Canti et al., Anal. Chem., 2007, 79:4666-4673) Reconstitute dried desalted sample in 20% acetonitrile/2% formic acid; 0.7E+07 0.6E+07 y t Wash TiO column with 20 µL 0.5% NH OH/40% acetonitrile, pH 10.5 i
2 4 s
n 0.5E+07 3957 e
t 3555
Equilibrate TiO column with 20 µL 20% acetonitrile/2% formic acid n 2 I 0.4E+07 3853 Load sample 3842 0.3E+07 29 Table IV. Phosphorylation Sites in Talin. Phosphorylation sites identified by the Protein Discovery Wash TiO2 column with 20 µL 80% acetonitrile/2% formic acid 192 3603 3481 Initiative are compared to data from PhosphoSITE and Phospho.ELM databases. Phosphorylation sites 0.2E+07 1720 3220 3205 183 279 627 854 1701 1743 2990 Wash TiO2 column with 5 µL 2% formic acid 533 945 1237 1245 2514 3243 are indicated in red; sites present in more than one database are highlighted in yellow. 1944 2226 2765 0.1E+07 Elute TiO2 column with 100 µL 200mM NH4HCO3, pH 9.0 into a C18 column 0 Wash C18 column with 2 mL 0.1M acetic acid (0.2 mL/min, 10 min) 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 Time (min) Start 2 hour gradient then begin data acquisition gi|1722836|sp|P42768|WASP_HUMAN (100%), 54451.5 Da Wiskott-Aldrich syndrome protein (WASp) 11 unique peptides, 39 unique spectra, 44 total spectra, 171/515 amino acids (33% coverage)
TiO + DHB enrichment method 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 2 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 Reconstitute dried desalted sample in 50 mg/mL DHB in 20% acetonitrile/2% formic acid; 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 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 Wash TiO2 column with 20 µL 0.5% NH4OH/40% acetonitrile, pH 10.5 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 Equilibrate TiO2 column with 20 µL 50 mg/mL DHB in 20% acetonitrile/2% formic acid 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 Load sample 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 Wash TiO2 column with 20 µL 50 mg/mL DHB in 20% acetonitrile/2% formic acid Figure 1. TIC from tryptic digest of WASP, C18 column, TiO2 enrichment and TiO2 + DHB enrichment Wash TiO2 column with 20 µL 80% acetonitrile/2% formic acid methods. Scaffold (Proteome Software Inc.) coverage map for each method is presented. Identified Wash TiO2 column with 5 µL 2% formic acid residues are displayed in yellow. Oxidized methionine and phosphorylated serine, threonine and tyrosine are displayed in green. Elute TiO2 column with 100 µL 200mM NH4HCO3, pH 9.0 into a C18 column Wash C18 column with 2 mL 0.1M acetic acid (0.2 mL/min, 10 min)
Start 2 hour gradient then begin data acquisition 1.6 Figure 2. Comparison of the two TiO TiO2 enrichment 2
t 1.4 enrichment methods. Approximately 1 n TiO2 + DHB enrichment u µg of a tryptic digest of Wiskott-Aldrich o 1.2 Summary C Syndrome Protein (WASP) was a r t 1 loaded onto a TiO column. TiO c 2 2 • Direct C18 analysis and TiO enrichment method combined typically provide more than 85%
e 2
p 0.8 enrichment was performed according Results S sequence coverage. If not, additional proteinases can be used to reach acceptable coverage (85%). to the different methods tested. Bound d
e 0.6 z peptides were eluted into a C18 i l
a column and subjected to LC-MS/MS • Addition of 2,5-dihydroxibenzoic acid (DHB) in the TiO2 enrichment method decreases the non-
m 0.4 r phosphorylated peptide background present in the sample. This method may be particularly useful in Table I. Results of different methods for phosphopetide identification using LC- o analysis. Peptide identification was MS/MS and SEQUEST analysis. N 0.2 performed using the SEQUEST the analysis of complex mixtures of proteins but is not of great utility in the identification of 0 algorithm. Spectra count results for phosphorylation site in isolated proteins.
Method Coverage Phosphopetides Total Peptides .Y .Q .R .Q s the four most abundant SR R K R ide S AV Q AV pt IT LE VM E pe PD G H GL ho QN QG LM G sp DI D GA DQ ho phosphopeptides and the sum of all TV FIE LV IE ll P • The methodologies established in the Protein Discovery Initiative allow for an expedious approach A *D G DF A C18 66% 15 380 GL IY *]E Y* *L .L *S LI LS K [S SK spectra counts of all phosphopeptides GP PQ ET to confidently identify phosphorylation sites in isolated proteins associated with cell migration. PV PP DA TiO enrichment 46% 49 102 P Q LT 2 GG AL Q Q SS EA are presented. Spectra count results GD PE IS G A AG HP PG R. PL .T are normalized to those observed TiO2 + DHB enrichment 33% 46 71 PL K LP GG • Future work will include stochiometric assessment of site specific phosphorylation using labeled R. using the TiO enrichment method. 2 and non-label methodologies.