Phosphotyrosine biased enrichment of tryptic peptides from cancer cells by combining py-mip and TiO 2 affinity resins

Transcription

1 Phosphotyrosine biased enrichment of tryptic peptides from cancer cells by combining py-mip and TiO 2 affinity resins Loreta Bllaci, Silje B. Torsetnes,, Celina Wierzbicka, Sudhirkumar Shinde, Börje Sellergren, Adelina Rogowska-Wrzesinska, and Ole N. Jensen, Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense, Denmark Department of Biomedical Sciences, Malmö University, Malmö Sweden Supporting Information Synthesis of py-mip 2 Preparation of the Semi-Complex Sample 2 Table S1. Composition of synthetic phosphopeptides 2 Implementation of Four Protocols for Phosphopeptides Enrichment in the Semi-Complex Sample 2 Figure S1. Schematics of the experimental workflow performed on a simple peptide mixture 3 TiO2 protocol for Phosphoenrichment of HeLa peptides 3 Figure S2. Relationship diagrams of phosphopeptides identifications among three highest yielding strategies 4 Figure S3. Effect of TiO 2 -py-mipenrichment on precursor ion intensities. 5 Statistical Analysis of Differences in Ion Intensities: TiO 2 vs. TiO 2 -py-mip 5 Figure S4: Numerical relationship between phosphopeptides identified minimally in 2 of 3 6 replicates, between TiO2, TiO2-pY-MIP and py-mip-tio2 Chemical Properties Analysis-Additional Statistical Data 6 Table S1. Summary of descriptive statistics (mean and standard deviation) of physicochemical properties of 7 phosphopeptides enriched by four protocols. 1

2 Synthesis of py-mip. Fmoc-pY-OEt (template, 0.4mmol) was dissolved in dry THF (4.8mL) followed by addition of pentamethylpiperidine (PMP, 0.8mmol) N-3, 5-bis (Trifluoromethyl)-phenyl-N-4- vinylphenylurea (functional monomer, 0.8mmol), acrylamide (co-monomer, 0.8mmol), pentaerythritol triacrylate (PETA, crosslinker, 10.4mmol) and 2, 2-Azo-bis (2, 4-dimethyl) valeronitrile (ABDV) (1% w/w of total monomer). The solution was transferred into a glass ampoule, previously cooled to 0 C, and purged dry nitrogen for 10 min. The ampoule was then flame-sealed and placed in water bath heated to 50 C. After 24 hours the monolith was lightly crushed and submitted to washing with acidified methanol (MeOH/0.1M HCl 80/20 v/v) and washing in Soxhlet apparatus with methanol for 24h. Next polymer was crushed and sieved to give particles in size between µm. Preparation of the Semi-Complex Sample. Twelve proteins were used to prepare the semi-complex mixture: Carbonic anhydrase (bovine), Ribonuclease B (bovine pancreas), Serum albumin (bovine), Lactoglobulin (bovine), Casein (bovine), Casein (bovine), Ovalbumin (chicken), Lysozyme (chicken), Alcohol dehydrogenase (Bakers yeast), Myoglobin (whale skeletal muscle), Amylase (Bacillus species) and Transferrin (human). All proteins were from Sigma-Aldrich except Transferrin which was from ACE Biosciences A/S. Of the twelve proteins, three were multiphosphorylated. Proteins were reduced, alkylated and digested by the following protocol: protein stocks, each with a concentration of 50 pmol/µl, were prepared in 50 mm triethylammonium bicarbonate buffer (TEAB). The proteins were reduced with 10 mm dithiothreitol (DTT), at room temperature for 30 min, and alkylated with 20 mm iodoacetamide (IAA) at room temperature, for 30 min, in dark conditions. Completeness of digestion of every protein was confirmed by MALDI-MS prior to being mixed and diluted with 0.1% TFA to a final concentration of 1 pmol/µl for each protein. Afterwards, a synthetic-peptides mixture was spiked in to a final concentration of 0.08 pmol/µl each peptide. The synthetic peptides mixture contained 12 peptides; four non-phosphorylated, four py and four ps phosphorylated (see Table S1 below). Table S2. Composition of synthetic phosphopeptides. Four synthetic phosphopeptides were ps-peptides (1-4), other four were py-peptides (5-8) and the last four (not listed) were regular (non-phosphorylated) peptides. Phosphopeptides sequence (M+H)+ 1 DRVpSIHPF VILGpSPAHR AVPSPPPApSPR WWWGSGPpSGSGGpSGGK DRVpYIHPF GADDSYpYTAAR GADDSpYpYTAR TRDIpYETDpYpYRK Implementation of Four Protocols for Phosphopeptide Enrichment in the Semi-Complex Sample. Four protocols: TiO2, py-mip, py-mip-tio2, and TiO2-pY-MIP, were tested on simple peptide mixture (concentration of the mixture was 1 pmol/µl), Figure S1. An aliquot corresponding to 1.25 pmol of untreated peptide mixture was used as control (Figure S1, A). Next, a volume of 5 µl (5 pmol) was divided and half was enriched with TiO2, while the other half was processed with py-mip, Figure S1, B-C. The procedure of TiO2 enrichment was performed similarly to the complex sample with the following modifications: the amount of TiO2 beads was reduced to 0.3 mg, the incubation of beads and sample was carried once for a duration of 10 minutes, and the volumes of the wash and elution solutions were also reduced to 50 µl. Also, the elution was carried in one step for 10 minutes. The resulting eluent containing phosphopeptides, was divided in two aliquots, 25 µl each, which were further dried by vacuum centrifuge. One dried aliquot was reconstituted in 1 µl matrix (20 mg/ml DHB, 1% PA) and analysed by MALDI MS, Figure S1, B1. The other sample was redissolved in 100 µl MIP loading buffer and processed with py-mip (Figure S1, B2) The procedure of py-mip was performed similarly to that of the complex mixture with the following changes: the volume of loading, wash and elution were reduced to 100, 25 and 100 µl (50 µl elution 1 and 50 µl elution 2, pooled) respectively. Also, the amount of py-mip packed in the column was reduced to 2 mg. The 2

3 centrifugation speed during the loading and wash were lowered to 800 rpmi. The eluent of TiO2-pY-MIP (Figure S1, B2) and half of py-mip (Figure S1, C1) were dried and reconstituted in MALDI matrix. The other half from py-mip was reconstituted in 100µL TiO2 loading buffer (Figure S1, C2). The rest of py-mip chromatography for this half was performed as described in the former paragraph. Figure S1. Schematics of the experimental workflow performed on a simple peptide mixture. The peptide mixture was spiked with twelve synthetic peptides, of which four py-peptides (sequences described in the previous page). The sample was processed following four analytical strategies: py-mip, TiO 2, TiO 2 -py-mip and py-mip-tio 2. An aliquot of the sample corresponding to 1.25 pmol was analysed by MALDI MS and the acquired spectrum was used as a reference of enrichment efficiency (A). For the experiment, a starting amount of mixture corresponding to 5 pmol was divided in two volumes and enriched with TiO 2 (B) and py-mip (C) Half of the eluents from these two processes were analysed by MALDI MS (B1, C1). The other two halves (1.25 pmol each) were further processed: the aliquot derived from TiO 2 underwent py- MIP (B2) while the aliquot derived from py-mip extraction was enriched with TiO 2 (C2) and finally the eluents were analysed by MALDI MS. TiO 2 Protocol for Phosphoenrichment of HeLa Peptides. Before use, TiO 2 beads were suspended in MeCN, aliquoted in low-binding vials and table-centrifuged for 30 seconds. The resulting supernatant (MeCN) was removed, leaving sedimented TiO 2 beads. Next, HeLa peptides aliquots (200 µg peptides determined by AAA) were then diluted to 1mL with loading buffer consisting of 80% MeCN, 5% TFA and 1M Glycolic acid, and added to the pellet of TiO 2 beads. Samples and beads were incubated twice, in high shaking conditions. For the first incubation, 1.2 mg TiO 2 beads per vial (peptide aliquot) were used. After incubation, vials were table-centrifuged, the supernatant was added to another vial with 0.6 mg of TiO 2 beads and subjected the second round of enrichment. The TiO 2 beads from two incubations were then pooled together with 100 µl loading buffer, vortexed for 30 seconds and table-centrifuged for 1 min. The flow-through were discarded, while the beads, were subjected to two rounds of washing. The first wash was performed in 100 µl of 80% MeCN in 1% TFA, vortexed for 30 sec and table-centrifuged for 1 min. The supernatant was discarded, while the beads were washed with 100 µl of 20% MeCN in 1% TFA. The supernatant was discarded again and the beads were incubated for 2 min in a vacuum centrifuge to accelerate evaporation of any trace of solvent left in the tube. The remaining bound entities were eluted from TiO 2 beads by sequential incubation with elution buffer consisting of 25% ammonia solution (ph 11, 6) in high shaking conditions for 20 min. The first eluent volume was 150 µl, and the second 30 µl. The two eluents were pooled and acidified prior to desalting. Desalted samples were then dried and stored at -20 C until further use. 3

4 Figure S2. Numerical relationship between phosphopeptides identifications among three highest yielding strategies. Venn diagrams illustrate: (A) the total identified phosphopeptides, (B) ps-, (C)pT- and (D)pY-peptides. For the site-specific Venn diagrams (B-D) only phosphopeptides phosphorylated on one type of residue were used. Diagrams were prepared based on the accumulated phosphopeptides in three replicates per method (n=3). 4

5 Figure S3. Effect of TiO 2 -py-mipenrichment on precursor ion intensities. For this experiment phosphopeptides which were commonly detected by TiO 2 -py-mip and TiO 2 were considered. Intensity refers to the average value of precursor ion area (PIA). Note that for one ps-peptide the value of the ratio was not plotted. Statistical Analysis of Differences in Ion Intensities: TiO 2 vs. TiO 2 -py-mip. Paired t-test result show no significant differences in the intensities of all types of phosphopeptides between TiO 2 - MIP and TiO 2 in favour of TiO 2. The results of three t-tests (of py, pt and ps) results are summarized are as follow: For py: P<0.0001, mean and SD of differences: M=-7.4 and SD =5.5. For ps: P<0.0001, mean and SD of differences: M=-7.6 and SD =7.1. For pt: P<0.0001, mean and SD of differences: M=-5.3 and SD =7.8 5

6 Figure S4: Numerical relationship between phosphopeptides identified minimally in 2 of 3 replicates, between TiO2, TiO2-pY-MIP and py-mip-tio2. Chemical Properties Analysis-Additional Statistical Data Basic Phosphopeptides. The py-mip and py-mip-tio 2 were statistically determined as extremely different from TiO 2 [(57±3 vs. 39±1%, P<0.001), and (52±3 vs. 39±1%, P<0.001), whilst the RF bars of TiO 2 -py-mip and TiO 2 were not considered statistically different (37±2 vs. 39±1%, P>0.05). Level of Phosphorylation and Peptides Length. Peptides with monophosphorylated sites derived from TiO 2 -py-mip, py-mip-tio 2 and py-mip had about 30% higher RF values than TiO 2 (94±1 vs. 59±2, P<0.001; 97±1 vs. 59±2, P<0.001 and 91±3 vs. 59±2, P<0.001). Also, there was a significantly higher proportion of short peptides (6-24 residues) than those enriched by TiO 2 (98± 1 vs. 76± 2, P 0.001, 90± 1 vs. 76±2, P<0.001, and 96±1 vs. 76±2 and P<0.001, respectively), Figure 4, lower panel. The opposite trend was detected for multiphosphorylated and long peptides ( 25 residues), Figure 4, lower panel. The relative prevalence of these peptides was shown to be lower for all py-mip-methods than for the TiO 2 reference method, and were all considered extremely significantly different. For the multiphosphorylated peptides, the approximate difference in RF value between all py-mip-methods against the reference was about 30-40% (TiO 2 -py-mip vs. TiO 2 : 6±1 vs. 41±2, P<0.001; py-mip-tio 2 vs. TiO 2 : 3±1 vs. 41±2, P<0.001, and py-mip vs. TiO 2 : 9±3 vs. 41±2, P<0.001). For the long peptides, the approximate difference in RF value between the TiO 2 -py-mip and py-mip approach against the reference was about 20%, while the py-mip-tio 2 was 10% (TiO 2 -py-mip vs. TiO 2 : 2±1 vs. 24±2, P<0.001; py-mip-tio 2 vs. TiO 2 : 11 ±2 vs. 24±2, P<0.001 and py-mip vs. TiO 2 : 3±2 vs. 24±2, P<0.001). 6

7 Table S3. Summary of descriptive statistics (mean and standard deviation) of physicochemical properties of phosphopeptides enriched by four protocols. 7