Addressing Trypsin Bias in Large Scale (Phospho)proteome Analysis by Size Exclusion Chromatography and Secondary Digestion of Large Post-Trypsin Peptides
Addressing Trypsin Bias in Large Scale (Phospho)proteome Analysis by Size Exclusion Chromatography and Secondary Digestion of Large Post-Trypsin Peptides
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DOI：
10.1021/pr100951t
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发表时间：
2011-02-01
影响因子：
4.4
通讯作者：
Quadroni, Manfredo
中科院分区：
文献类型：
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作者：
Tran, Bao Quoc;Hernandez, Celine;Quadroni, Manfredo
在绝大多数自下而上的蛋白质组学研究中，仅使用哺乳动物胰蛋白酶进行蛋白质消化。尽管胰蛋白酶显然是最好的酶，但单独使用胰蛋白酶很少导致完全的序列覆盖，即使对于丰富的蛋白质也是如此。通常认为这是因为许多胰蛋白酶肽太短或太长而无法通过RPLC-MS/MS鉴定。我们通过计算机分析表明，三种蛋白质组的总序列的20-30%（粟酒裂殖酵母，酿酒酵母，和智人）预计将被大胰蛋白酶后肽（LpTPs）与M-r大于3000 Da。然后，我们建立了尺寸排阻色谱法，以基于尺寸将复合酵母胰蛋白酶消化酶破碎成肽池。我们发现，LpTP的二次消化，然后进行LC-MS/MS分析，导致所鉴定的蛋白质的显著增加，并且与单独的胰蛋白酶消化相比，平均序列覆盖率相对增加32-50%。应用该方法分析了S. pombe和人细胞系鉴定了显著部分的新磷酸化位点。总的来说，我们的数据表明，特异性靶向LpTPs可以补充标准的自下而上的工作流程，以揭示蛋白质组中很大程度上被忽视的部分。
In the vast majority of bottom-up proteomics studies, protein digestion is performed using only mammalian trypsin. Although it is clearly the best enzyme available, the sole use of trypsin rarely leads to complete sequence coverage, even for abundant proteins. It is commonly assumed that this is because many tryptic peptides are either too short or too long to be identified by RPLC-MS/MS. We show through in silico analysis that 20-30% of the total sequence of three proteomes (Schizosaccharomyces pombe, Saccharomyces cerevisiae, and Homo sapiens) is expected to be covered by Large post-Trypsin Peptides (LpTPs) with M-r above 3000 Da. We then established size exclusion chromatography to fractionate complex yeast tryptic digests into pools of peptides based on size. We found that secondary digestion of LpTPs followed by LC-MS/MS analysis leads to a significant increase in identified proteins and a 32-50% relative increase in average sequence coverage compared to trypsin digestion alone. Application of the developed strategy to analyze the phosphoproteomes of S. pombe and of a human cell line identified a significant fraction of novel phosphosites. Overall our data indicate that specific targeting of LpTPs can complement standard bottom-up workflows to reveal a largely neglected portion of the proteome.