Automated platform of µlc-ms/ms using SAX trap column for

Transcription

1 Analytical and Bioanalytical Chemistry Electronic Supplementary Material Automated platform of µlc-ms/ms using SAX trap column for highly efficient phosphopeptide analysis Xionghua Sun, Xiaogang Jiang 1

2 Experimental Section 1 Materials and Reagents. Magic C18 AQ (5 μm, 100 and 200 Å pore) was purchased from Michrom BioResources (Auburn, CA), and strong anion exchange (5 μm, 120 Å pore) was from Thermo Inc (San Jose, CA). Fused-silica capillaries (50, 75 and 100 μm I.D.) were bought from Polymicro Technologies (Phoenix, AZ). All the water used in the experiment was purified using a Mill-Q system (Millipore, Bedford, MA). Trypsin was from Promega (Madison, WI). 2 Sample Preparation. Mouse liver were homogenized in lysis buffer consisting of 8 M urea, 4% CHAPS (w/v), 65 mm DTT, 1 mm EDTA, 0.5 mm EGTA, and a mixture of phosphatase inhibitors (1 mm PMSF, 0.2 mm Na 3 VO 4, 1 mm NaF) in 40 mm Tris-HCl at ph 7.4. After centrifuged at g for 1 h, the supernatant was collected. The protein in the supernatant was precipitated by acetone at -20 o C, and the precipitate was redissolved in the solution (8 M urea, 100 mm ammonium bicarbonate, ph 8.3). The protein sample was reduced by DTT and alkylated by iodoacetamide. Then the solution was diluted to 1 M urea, and the ph value was adjusted to 8.1. Finally, trypsin was added (trypsin:protein, 1:50) and incubated at 37 o C for 20 h. The phosphopeptides were enriched from the protein digest as follows. Briefly, 100 ug of protein digest was mixed with 100 μl ZrO 2 nanoparticles suspension in 10% HAc/50% ACN (30 mg/ml). The resulting solution was incubated for 30 min, and it was then centrifuged at g for 5 min. After the removal of supernatant, the nanoparticles were rinsed with 100 μl solution of 10% HAc/50% ACN to remove 2

3 non-specifically adsorbed peptides. The trapped phosphopeptides on ZrO 2 nanoparticles were eluted using 12.5 % NH 3 H 2 O under the sonication for 10 min. After centrifugation, the supernatant was collected as phosphopeptide sample and lyophilized to dryness. 3 Automated µlc-ms/ms system The LC-MS/MS system consisted of a quaternary Surveyor pump, a Surveyor autosampler and an LTQ linear ion trap mass spectrometer equipped with a nanospray source and a six-port/two-position valve (Thermo, San Jose, CA). The four buffer solutions used for the quaternary pump were 0.1% formic acid (buffer A), 99.9% ACN/0.1% formic acid (buffer B), 10 mm NH 4 Ac (buffer C, ph 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, respectively), and 500 mm NH 4 Ac, ph 2.5 (buffer D). The configuration for automated sample injection using SAX trap column was shown in Figure 1. The trap column with I.D. of 100 μm was prepared by packing 2 cm length of SAX resin into the capillary with frit. The analytical column with I.D. of 75 μm was packed with C18 particles. The procedure of automated µhplc-ms/ms analysis of phosphopeptides using the SAX trap column had following four steps: 1) Equilibrating the SAX trap column system. The SAX trap column system was equilibrated by flushing with buffer C at flow of 150 µl/min for 10 min (The valve position was switched and the venting capillary was blocked). 2) Loading phosphopeptide sample onto the SAX trap column. Phosphopeptide sample (25 μl) was introduced onto the trap column at flow of 2 μl/min for 30 min (The valve position was switched and the splitting capillary was blocked). The mobile phase used was the same as that of step 1. 3) Eluting 3

4 sample from SAX trap column onto C18 analytical column. The phosphopeptide retained on SAX trap column was eluted onto the analytical column by flushing with a buffer containing low ph (buffer A) or high salt (buffer D) buffer utilizing a split flow of 200 nl/min for 5 min (The valve position was switched and the venting capillary was blocked). 4) Gradient separation. The system was first equilibrated with buffer A for 10 min, and then separation of phosphopeptides was achieved by using a binary solvent composition gradient developed with buffer A and buffer B from 5% to 35% buffer B for 60 min, and from 35% to 80% for 10 min. For the system using C18 trap column, after the sample was loaded with 0.1% HAc onto the C18 trap column, a gradient directly started for the separation. The LTQ instrument was operated at positive ion mode. The temperature of the ion-transfer capillary was set at 200 o C. A voltage of 1.8 kv was applied to the cross and the normalized collision energy was set at 35.0%. The detection of phosphopeptides was performed in which the mass spectrometer was set as a full scan MS followed by five data-dependant MS/MS (MS 2 ). Subsequently acquisition of MS/MS/MS (MS 3 ) spectra was automatically triggered when the three most intense peak from the MS/MS spectrum corresponded to a neutral loss event of 98, 49 and 33±1 Da for the precursor ion with 1+, 2+, 3+ charge states, respectively. The dynamic exclusion function was set as follows: repeat count 2, repeat duration 30 s, and exclusion duration 90 s. 4 Database searching and data analysis The database searching and data analysis are the same as previous report[1-2]. 4

5 All MS 2 and MS 3 spectra were searched against the non-redundant mouse protein database of Mouse International Protein Index (ipi.mouse.3.21.fasta) by Bioworks 3.2. The following parameters were set for the search, precursor-ion mass tolerance, 2 Da; fragment-ion mass tolerance, 1 Da; missed cleavages, 2; static modification, Cys (+57). For the search with MS 2 data, dynamic modifications were set for oxidized Met (+16), and phosphorylated Ser, Thr and Tyr (+80). For the searching with MS 3 data, besides above set, dynamic modifications were also set for water loss on Ser, Thr (-18). Criteria used for filtering data derived from MS 2 spectra were: Cross-correlation value (Xcorr) larger than 1.0, 1.5, and 2.0 used for singly, doubly, and triply charged ions respectively,and Delta Cn values are not less than 0.1; while the criteria used for filtering data derived form MS 3 spectra were: Xcorr larger than 1.0 used for all charged ions,and Delta Cn values are not less than All output results were combined together using homemade software to get the list of phosphopeptides which were derived both from MS 2 and MS 3. Manual validation was further carried out for peptides passing the above criteria. Criteria used for manual validation included: (a) MS 2 must be followed by MS 3,the peptides sequence derived from MS 2 and MS 3 spectra must be same and in one MS cycle;(b) if the same scan has two or more sequences, delete the sequences which have lower, keep the sequence with the highest Xcorr; (c) if the same spectral have the same sequence but different phosphorylated sites, delete the lower one, keep the sites with the highest Xcorr; (d) the phosphoric acid neutral loss peak to phosphoserine and phosphothreonine must be the top three peak; (e) the spectrum must be of good quality with fragment ion clearly above the 5

6 baseline noise in either of MS 2 or MS 3 spectra; (f) sequential members of the b- or y- series were observable in either of MS 2 or MS 3 spectra. References: 1. Jiang X, Han G, Feng S, Ye M, Yao X, Zou H (2008) Automatic validation of phosphopeptide identifications by the MS2/MS3 target-decoy search strategy. Journal of proteome research 7 (4): doi: /pr700675j 2. Ulintz PJ, Yocum AK, Bodenmiller B, Aebersold R, Andrews PC, Nesvizhskii AI (2009) Comparison of MS(2)-only, MSA, and MS(2)/MS(3) methodologies for phosphopeptide identification. Journal of proteome research 8 (2): doi: /pr800535h 6