A challenge for shotgun proteomics is the identification of low abundance proteins, which is always hampered owing to the extreme complexity of protein digests and highly dynamic concentration range of proteins. To reduce the complexity of the peptide mixture, we developed a novel method to selectively enrich N-terminal proline peptides via hydrazide chemistry. This method consisted of ortho-phthalaldehyde (OPA) blocking of primary amines in peptides, reductive glutaraldehydation of N-terminal proline and solid phase hydrazide chemistry enrichment of aldehyde-modified N-terminal proline peptide. After enrichment, the number of detected peptides containing N-terminal proline increased from 1304 to 4039 and the ratio of N-terminal proline peptides jumped from 4.4% to 93.7%, showing good enrichment specificity towards N-terminal proline peptides. Besides, the ratio of identified peptides to proteins was decreased from 7.8 (29751/3811) to 1.5 (4347/2821), indicating that sample complexity was drastically reduced through this method. As a result, this novel approach for enriching N-terminal proline peptides is effective in identification of low abundance protein owing to the reduction of sample complexity.

中文翻译：

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通过酰肼化学选择性富集 N 端脯氨酸肽用于蛋白质组学分析

霰弹枪蛋白质组学的一个挑战是低丰度蛋白质的鉴定，由于蛋白质消化的极端复杂性和蛋白质的高度动态浓度范围，这总是受到阻碍。为了降低肽混合物的复杂性，我们开发了一种通过酰肼化学选择性富集 N 端脯氨酸肽的新方法。该方法包括邻苯二甲醛 (OPA) 阻断肽中的伯胺、N 端脯氨酸的还原戊二醛和醛修饰的 N 端脯氨酸肽的固相酰肼化学富集。富集后检测到的含N端脯氨酸肽段从1304个增加到4039个，N端脯氨酸肽段比例从4.4%跃升至93.7%，对N端脯氨酸肽段具有良好的富集特异性。此外，鉴定的肽与蛋白质的比率从 7.8 (29751/3811) 降低到 1.5 (4347/2821)，表明通过这种方法大大降低了样品的复杂性。因此，由于降低了样品复杂性，这种富集 N 端脯氨酸肽的新方法可有效识别低丰度蛋白质。