Keough T, Lacey M P, Youngquist R S
The Procter and Gamble Company, Miami Valley Laboratories, PO Box 538707, Cincinnati, OH 45253-8707, USA.
Rapid Commun Mass Spectrom. 2002;16(11):1003-15. doi: 10.1002/rcm.670.
Solid-phase sulfonation of tryptic peptides adsorbed to C18 muZipTips has been carried out to facilitate de novo sequencing with mass spectrometry. Peptides are reacted with the sulfonation reagent while they are still adsorbed to the solid phase. Excess reagent passes through the ZipTip to waste. Washing the products before subsequent elution from the mini-column also affords sample cleanup prior to analysis. Near quantitative N-terminal sulfonation can be achieved reliably at room temperature in only a few seconds. The method has been applied successfully to model peptides and to solution or in-gel digests of proteins. Current sequencing limits are about 100 fmol of protein. Multiplexed sample sulfonation reactions have been carried out with a manual 8-position micropipettor or using centrifugal force to reliably pass reagents and wash solutions over sample-loaded ZipTips. With multiplexing, overall preparation times have been reduced to about 1 min per sample. The solid-phase format facilitates efficient use of precious digest samples by enabling them to be recovered from the matrix-assisted laser desorption/ionization (MALDI) sample stage after mass fingerprinting, derivatized and re-analyzed by MALDI postsource decay mass spectrometry.
对吸附于C18 μZipTip上的胰蛋白酶肽段进行了固相磺化，以促进质谱法的从头测序。肽段在仍吸附于固相时与磺化试剂反应。过量试剂通过ZipTip进入废液。在从小型柱中进行后续洗脱之前洗涤产物，也能在分析前对样品进行净化。在室温下仅需几秒钟就能可靠地实现近定量的N端磺化。该方法已成功应用于模拟肽段以及蛋白质的溶液消化物或胶内消化物。目前的测序极限约为100 fmol蛋白质。已使用手动8通道微量移液器或利用离心力可靠地使试剂和洗涤溶液通过加载有样品的ZipTip，进行了多重样品磺化反应。通过多重反应，每个样品的总体制备时间已缩短至约1分钟。固相形式有助于有效利用珍贵的消化样品，使它们能够在进行质量指纹分析后从基质辅助激光解吸/电离（MALDI）样品台上回收，进行衍生化处理，然后通过MALDI源后衰变质谱法重新分析。
