基质辅助激光解析飞行时间质谱分析条件及准确度对蛋白质数据库搜索结果的影响

Effect of Matrix Assisted Laser Desorption Ionization-Time of Figlt-Mass Spectrometric Analysis Conditions and Accuracy Rating on Protein Database Search in Peptide Mass Fingerprint Identify Proteins

考察了基质辅助激光解析飞行时间质谱获得肽质量指纹谱(PMF)时主要分析条件及准确度对蛋白质数据库搜索结果的影响。将牛血清白蛋白(BSA)经聚丙烯酰胺凝胶电泳(SDS-PAGE)分离、酶解;肽段经质谱分析得PMF数据;用MS-Fit引擎在相同数据库及参数下搜索。得出:(1)激光脉冲击打次数在300次以前时,蛋白序列和肽段覆盖率逐步增加;当击打400次时,覆盖率呈下降趋势;(2)随着激光强度增加,目标蛋白得分、氨基酸序列和肽段的覆盖率逐步增加,但当强度增到1413时,则呈现降低趋势;(3)随延迟时间的增加,肽覆盖率呈递增趋势,但当达260ns后呈下降趋势。按照优化的分析条件对同一位点重复3次,重复性良好。通过对质谱数据准确度的考察,发现以Mix2为外标,数据跟标准值差异越小时,则经其校正后的肽段数据所搜索目标蛋白的得分越高、排序靠前、检出预选蛋白个数越少,可信度越高;反之,难获得候选蛋白。结果表明:质谱准确度和分析条件对蛋白质数据库搜索影响显著。

To inspect effect of main MS analysis conditions and data accuracy that through MALDI-TOF-MS to obtain peptides mass fingerprint（PMF） for protein search results, BSA had been separated by SDS-PAGE, hydrolyzed by trypsin, identified by MS and the PMF data would be searched through MS-Fit search engine. The results showed that sequence coverage and peptides coverage gradually increase when laser strike numbers under 300, strike number at 400, which appeared to descend tendency. Target protein score, amino acid se- quence and peptides coverage all appeared to increase follow laser intensity increase, when laser intensity at 1413, which showed descend tendency. Peptides coverage showed increasing tendency follow delay time increasing, when delay time at 260 ns, which took on descend. Based on optimized analysis conditions and checked the same site for 3 times, the result showed that reproducibility was better. To inspected accuracy of MS data, Mix 2 was adopted as external standard to remedy MS primitive data, we found score more higher, the order ahead, detected numbers fewer and the reliability better as the MS data near standard value. In contrast, we almost didnt get target protein. The results showed that MS accuracy and analysis conditions existed in distinguished influence on protein database search.