PO0020 Enhanced Recovery of Trypsin Digested Proteins Using Dispersive Pipette Extraction for Downstream Proteomic Analysis
來源:IMCS
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作者:YuzheNie1,PongkwanSitasuwan2, L. Andrew Lee2, QianWang1
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發布時間: 970天前
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962 次瀏覽
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INTRODUCTION
For clinical proteomics, targeted peptide extraction and desalting efficiencies areessential to obtaining high quality mass spectra data. In this study, we report anovel peptide purification method usingIMCStips?,a disposable pipetteextraction tip with sorbent loosely contained inside a pipette tip. Using bovineserum albumin as a model protein and four other human serum proteins,trypsin-digestedsamples were readilydesaltedusingIMCStips? with reversephase resin. The purificationimproved signalintensities, signal-to-noise ratiosand sequencecoverage in comparison to other commercially available tips.Sincethis method can be effortlessly adapted to an automated online format, weenvision it to be applied in the high throughput clinical proteomics application.
For clinical proteomics, targeted peptide extraction and desalting efficiencies areessential to obtaining high quality mass spectra data. In this study, we report anovel peptide purification method usingIMCStips?,a disposable pipetteextraction tip with sorbent loosely contained inside a pipette tip. Using bovineserum albumin as a model protein and four other human serum proteins,trypsin-digestedsamples were readilydesaltedusingIMCStips? with reversephase resin. The purificationimproved signalintensities, signal-to-noise ratiosand sequencecoverage in comparison to other commercially available tips.Sincethis method can be effortlessly adapted to an automated online format, weenvision it to be applied in the high throughput clinical proteomics application.
Four purifiedhuman proteinsamples(albumin,prealbumin, alpha-1-acidglycoprotein,transferrin)were purchased from Athens Research and Technology (Athens, GA). Bovine serumalbumin (BSA)was purchased from Fisher Scientific.AcclaimTM120, C18, 5 μm (4.6×100 mm) from Thermo Scientific was used for peptide separation on UPLC with a diode array detector (Vanquish?, Thermo Scientific). All solvents were mass spectrometry gradepurchased from Fisher Scientific. Sequencing grade trypsin was purchased fromSigma Aldrich.IMCStipswere provided by the vendor.Ziptips? (Millipore) andPierce C18 tips were purchased from Fisher Scientific.
Initial workflow validation studies and product comparison studies were conducted using 0.01 mg/mLBSA in10mMPBS (phosphate buffered saline) and 0.1% TFA (trifluoroaceticacid) Ziptip?purification-followed the vendor’s suggested method using 10 μL of 0.01mg/mL BSA and eluted with 5 μL 50% acetonitrile/0.1% TFA;
Pierce?C18tippurification-followed the vendor’s suggested method using 100μLof 0.01mg/mLBSAand eluted with 50 μL 50% acetonitrile/0.1% TFA;
IMCStipspurificationusing following protocol:
1.Condition the tip with 100 μL acetonitrile, repeat once;
2.Equilibrate by aspirating 100 μL water/0.1% TFA, repeat once;
3.Aspirate 100 μL of sample, dispense and aspirate sample for 3 cycles;
4.Rinse the tip by aspirating 100 μL water/0.1% TFA and discarding solvent,repeat once;
5.Elute the sample with 50 μL 50%acetonitrile/0.1% TFA, aspirate and dispense 3 cycles
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Initial workflow validation studies and product comparison studies were conducted using 0.01 mg/mLBSA in10mMPBS (phosphate buffered saline) and 0.1% TFA (trifluoroaceticacid) Ziptip?purification-followed the vendor’s suggested method using 10 μL of 0.01mg/mL BSA and eluted with 5 μL 50% acetonitrile/0.1% TFA;
Pierce?C18tippurification-followed the vendor’s suggested method using 100μLof 0.01mg/mLBSAand eluted with 50 μL 50% acetonitrile/0.1% TFA;
IMCStipspurificationusing following protocol:
1.Condition the tip with 100 μL acetonitrile, repeat once;
2.Equilibrate by aspirating 100 μL water/0.1% TFA, repeat once;
3.Aspirate 100 μL of sample, dispense and aspirate sample for 3 cycles;
4.Rinse the tip by aspirating 100 μL water/0.1% TFA and discarding solvent,repeat once;
5.Elute the sample with 50 μL 50%acetonitrile/0.1% TFA, aspirate and dispense 3 cycles
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