Rapid identification of comigrating gel-isolated proteins by ion trap mass spectrometry
Rapid identification of comigrating gel-isolated proteins by ion trap mass spectrometry
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DOI：
10.1002/elps.1150190612
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发表时间：
1998-05-01
期刊：
影响因子：
2.9
通讯作者：
Stults, JT
中科院分区：
文献类型：
--
作者：
Arnott, D;Henzel, WJ;Stults, JT
为了寻找新的核结合蛋白，我们分析了十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）凝胶的两条条带，发现每条条带都含有许多蛋白质，随后在四极离子阱仪器上通过串联质谱法（MS/MS）进行了鉴定。在电印迹转移后，用胰蛋白酶在聚偏二氟乙烯（PVDF）膜上原位消化这些条带。基质辅助激光解吸/电离-质谱法（MALDI-MS）对每种肽混合物的2.5%进行分析，然后对肽质量进行初始数据库搜索，未能识别出蛋白质。多肽通过反相毛细管高效液相色谱（HPLC）分离，以预测随后的Edman降解，但通过MALDI-MS对色谱组分进行质谱分析发现存在多个共溶肽，这使得该方法无法使用。所选组分采用毛细管高效液相色谱-电喷雾离子阱质谱法进行分析。串联质谱法提供了重要的片段，从中可以推断出许多肽的全部或部分序列。在一个病例中使用两个阶段的碎片（MS3）来确定附加序列。数据库检索，每次使用单个肽质量加上部分序列，从45 kDa的单个电泳带中鉴定出4个蛋白质，从60 kDa的第二个带中鉴定出4个蛋白质。其中许多蛋白质来源于人类角蛋白。在MALDI-MS光谱中存在多个匹配的肽团，证实了蛋白质鉴定。此外，通过对MS/MS数据的解释，确定了一个在蛋白质或DNA数据库中未发现的新序列。这些结果证明了四极离子阱在混合物中识别多种蛋白质和重新确定肽序列方面的能力。用改进的数据库搜索算法对片段数据进行重新分析，结果表明，在没有质谱解释或氨基酸序列的情况下，从有限数量的片段离子质量中鉴定出了相同的蛋白质组。讨论了仅从片段离子质量进行蛋白质鉴定的意义，包括低信号水平、减少必要的解释专业知识和提高速度的优点。
In the search for novel nuclear binding proteins, two bands from a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel were analyzed and each was found to contain a number of proteins that subsequently were identified by tandem mass spectrometry (MS/MS) on a quadrupole ion trap instrument. The bands were digested with trypsin in situ on a polyvinylidene difluoride (PVDF) membrane following electroblot transfer. Analysis of a 2.5% aliquot of each peptide mixture by matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) followed by an initial database search with the peptide masses failed to identify the proteins. The peptides were separated by reversed-phase capillary high performance liquid chromatography (HPLC) in anticipation of subsequent Edman degradation, but mass analysis of the chromatographic fractions by MALDI-MS revealed multiple, coeluting peptides that precluded this approach. Selected fractions were analyzed by capillary HPLC-electrospray ionization-ion trap mass spectrometry. Tandem mass spectrometry provided significant fragmentation from which full or partial sequence was deduced for a number of peptides. Two stages of fragmentation (MS3) were used in one case to determine additional sequence. Database searches, each using a single peptide mass plus partial sequence, identified four proteins from a single electrophoretic band at 45 kDa, and four proteins from a second band at 60 kDa. Many of these proteins were derived from human keratin. The protein identifications were corroborated by the presence of multiple matching peptide masses in the MALDI-MS spectra. In addition, a novel sequence, not found in protein or DNA databases, was determined by interpretation of the MS/MS data. These results demonstrate the power of the quadrupole ion trap for the identification of multiple proteins in a mixture, and for de novo determination of peptide sequence. Reanalysis of the fragmentation data with a modified database searching algorithm showed that the same sets of proteins were identified from a limited number of fragment ion masses, in the absence of mass spectral interpretation or amino acid sequence. The implications for protein identification solely from fragment ion masses are discussed, including advantages for low signal levels, for a reduction of the necessary interpretation expertise, and for increased speed.