PO0020 Enhanced Recovery of Trypsin Digested Proteins Using Dispersive Pipette Extraction for Downstream Proteomic Analysis
来源:IMCS
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作者:YuzheNie1,PongkwanSitasuwan2, L. Andrew Lee2, QianWang1
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发布时间: 1714天前
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1862 次浏览
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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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