Size-Sorting Combined with Improved Nanocapillary Liquid Chromatography-Mass Spectrometry for Identification of Intact Proteins up to 80 kDa

作者：Vellaichamy Adaikkalam; Tran John C; Catherman Adam D; Lee Ji Eun; Kellie John F; Sweet Steve M M; Zamdborg Leonid; Thomas Paul M; Ahlf Dorothy R; Durbin Kenneth R; Valaskovic Gary A; Kelleher Neil L^*

来源：Analytical Chemistry, 2010, 82(4): 1234-1244.

DOI：10.1021/ac9021083

Despite the availability of ultra-high-resolution mass spectrometers, methods for separation and detection of intact proteins for proteome-scale analyses are still in a developmental phase. Here we report robust protocols for online LC-MS to drive high-throughput top-down proteomics in a fashion similar to that of bottom-up proteomics. Comparative work on protein standards showed that a polymeric stationary phase led to superior sensitivity over a silica-based medium in reversed-phase nanocapillary LC, with detection of proteins >50 kDa routinely accomplished in the linear ion trap of a hybrid Fourier transform mass spectrometer. Protein identification was enabled by nozzle-skimmer dissociation and detection of fragment ions with <10 ppm mass accuracy for highly specific database searching using tailored software. This overall approach led to identification of proteins up to 80 kDa, with 10-60 proteins identified in single LC-MS runs of samples from yeast and human cell lines prefractionated by their molecular mass using a gel-based sieving system.

• 出版日期2010-2-15