Reliable detection of deamidated peptides from lens crystallin proteins using changes in reversed-phase elution times and parent ion masses
Reliable detection of deamidated peptides from lens crystallin proteins using changes in reversed-phase elution times and parent ion masses
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DOI：
10.1021/pr070182x
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发表时间：
2007-09-01
影响因子：
4.4
通讯作者：
David, Larry L.
中科院分区：
文献类型：
--
作者：
Dasari, Surendra;Wilmarth, Phillip A.;David, Larry L.
识别脱酰胺肽使用低分辨率质谱是困难的，因为传统的数据库搜索程序不能准确地检测到修饰的肽时，质量差只有0.984 Da。在这项研究中，我们利用差分反相洗脱行为的脱酰胺和相应的未修饰的肽形式，以显着提高脱酰胺检测低分辨率LCQ离子阱仪器。我们还通过平均每个色谱峰的调查扫描来改进未修饰和脱酰胺肽形式的质量测量。使用一系列正常（3天大、2岁、18岁、35岁和70岁）和白内障（93岁）人透镜样品的胰蛋白酶消化来产生大量潜在脱酰胺肽。通过强阳离子交换（SCX）色谱法，然后通过反相（RP）色谱法分离复杂的肽混合物。使用合成肽显示未修饰和脱酰胺肽在SCX分离过程中共洗脱，并在所用RP条件下完全分离。对于70年老化的透镜样品，手动测定脱酰胺透镜肽及其相应未修饰形式的保留时间偏移（RTS）和质量差（AM）。这些值用于指定SEQUEST检索中正确或不正确的脱酰胺鉴定，其中脱酰胺被指定为变量修饰。人工验证合成肽、3天和70年样品的SEQUEST鉴定具有总体42%的脱酰胺检测准确度。使用RTS和AM约束过滤SEQUEST鉴定导致>93%的脱酰胺检测准确度。开发了一种算法来自动化该方法，并检测到72个晶体蛋白脱酰胺位点，其中18个先前未在人透镜组织中报告。
Identifying deamidated peptides using low-resolution mass spectrometry is difficult because traditional database search programs cannot accurately detect modified peptides when the mass differences are only 0.984 Da. In this study, we utilized differential reversed-phase elution behavior of deamidated and corresponding unmodified peptide forms to significantly improve deamidation detection on a low-resolution LCQ ion trap instrument. We also improved the mass measurements of unmodified and deamidated peptide forms by averaging survey scans across each chromatogram peak. Tryptic digests of a series of normal (3-day old, 2-year old, 18-year old, 35-year old, and 70-year old) and cataractous (93-year old) human lens samples were used to produce large numbers of potentially deamidated peptides. The complex peptide mixtures were separated by strong cation exchange (SCX) chromatography followed by reversed-phase (RP) chromatography. Synthetic peptides were used to show that unmodified and deamidated peptides coeluted during the SCX separation and were completely resolved with the RP conditions used. Retention time shifts (RTS) and mass differences (AM) of deamidated lens peptides and their corresponding unmodified forms were manually determined for the 70-year old lens sample. These values were used to assign correct or incorrect deamidation identifications from SEQUEST searches where deamidation was specified as a variable modification. Manual validation of SEQUEST identifications from synthetic peptides, 3-day old, and 70-year old samples had an overall 42% deamidation detection accuracy. Filtering SEQUEST identifications using RTS and AM constraints resulted in >93% deamidation detection accuracy. An algorithm was developed to automate this method, and 72 Crystallin deamidation sites, 18 of which were not previously reported in human lens tissue, were detected.