Online protein digestion in membranes between capillary electrophoresis and mass spectrometry
Online protein digestion in membranes between capillary electrophoresis and mass spectrometry
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DOI：
10.1039/d3an00106g
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发表时间：
2023-03-08
期刊：
影响因子：
4.2
通讯作者：
Bruening，Merlin L.
中科院分区：
文献类型：
--
作者：
Ryan，Kendall A.;Bruening，Merlin L.
本研究采用含胃蛋白酶的膜在线消化蛋白质后，毛细管电泳（CE）分离和串联质谱法之前。分离后的蛋白水解允许来自给定蛋白质的肽以单个塞进入质谱仪。因此，迁移时间可以作为确认肽鉴定的额外标准。膜位于鞘流电喷雾电离（ESI）源中，以便在喷雾进入质谱仪之前立即进行消化，从而限制消化肽的分离。使用相同的膜，在10小时内进行的20次连续CE分析期间，消化可重复发生。此外，在用CE分离六种未还原蛋白质的混合物后，在线消化促进了所有蛋白质的至少2种可识别肽的蛋白质识别。肌红蛋白和碳酸酐酶II的序列覆盖率>75%，但含有二硫键的蛋白质的序列覆盖率要低得多。开发用于有效分离还原蛋白质或鉴定交联肽的方法应增强具有二硫键的蛋白质的序列覆盖率。从特定蛋白质鉴定的肽的迁移时间相差<1030 s，这允许拒绝一些假肽鉴定。
This research employs pepsin-containing membranes to digest proteins online after a capillary electrophoresis (CE) separation and prior to tandem mass spectrometry. Proteolysis after the separation allows the peptides from a given protein to enter the mass spectrometer in a single plug. Thus, migration time can serve as an additional criterion for confirming the identification of a peptide. The membrane resides in a sheath-flow electrospray ionization (ESI) source to enable digestion immediately before spray into the mass spectrometer, thus limiting separation of the digested peptides. Using the same membrane, digestion occurred reproducibly during 20 consecutive CE analyses performed over a 10 h period. Additionally, after separating a mixture of six unreduced proteins with CE, online digestion facilitated protein identification with at least 2 identifiable peptides for all the proteins. Sequence coverages were >75% for myoglobin and carbonic anhydrase II but much lower for proteins containing disulfide bonds. Development of methods for efficient separation of reduced proteins or identification of cross-linked peptides should enhance sequence coverages for proteins with disulfide bonds. Migration times for the peptides identified from a specific protein differed by <∼30 s, which allows for rejection of some spurious peptide identifications.