De Novo Sequencing of Unique Sequence Tags for Discovery of Post-Translational Modifications of Proteins

De novo sequencing is a spectrum analysis approach for mass spectrometry data to discover post-translational modifications in proteins; however, such an approach is still in its infancy and is still not widely applied to proteomic practices due to its limited reliability. In this work, we describe a de novo sequencing approach for the discovery of protein modifications based on identification of the proteome UStags (Shen, Y.; Tolic?, N.; Hixson, K. K.; Purvine, S. O.; Pasa?-Tolic?, L.; Qian, W. J.; Adkins, J. N.; Moore, R. J.; Smith, R. D. Anal. Chem. 2008, 80, 1871?1882). The de novo information was obtained from Fourier-transform tandem mass spectrometry data for peptides and polypeptides from a yeast lysate, and the de novo sequences obtained were selected based on filter levels designed to provide a limited yet high quality subset of UStags. The DNA-predicted database protein sequences were then compared to the UStags, and the differences observed across or in the UStags (i.e., the UStags�� prefix and suffix sequences and the UStags themselves) were used to infer possible sequence modifications. With this de novo?UStag approach, we uncovered some unexpected variances within several yeast protein sequences due to amino acid mutations and/or multiple modifications to the predicted protein sequences. To determine false discovery rates, two random (false) databases were independently used for sequence matching, and ��3% false discovery rates were estimated for the de novo?UStag approach. The factors affecting the reliability (e.g., existence of de novo sequencing noise residues and redundant sequences) and the sensitivity of the approach were investigated and described. The combined de novo?UStag approach complements the UStag method previously reported by enabling the discovery of new protein modifications.

从头测序（de novo sequencing）是一种针对质谱数据的光谱分析方法，用于发现蛋白质中的翻译后修饰（post-translational modifications）；然而此类方法仍处于起步阶段，且因可靠性有限，尚未广泛应用于蛋白质组学（proteomics）实践。本研究中，我们提出一种基于蛋白质组UStags（UStags）识别的蛋白质修饰发现从头测序方法（参考文献：Shen, Y.; Tolić, N.; Hixson, K. K.; Purvine, S. O.; Pasa?-Tolić, L.; Qian, W. J.; Adkins, J. N.; Moore, R. J.; Smith, R. D. Anal. Chem. 2008, 80, 1871–1882）。该研究从酵母裂解液（yeast lysate）来源的肽与多肽的傅里叶变换串联质谱（Fourier-transform tandem mass spectrometry）数据中获取从头测序信息，并基于预设过滤条件筛选所获序列，以得到规模有限但质量优异的UStags子集。随后将DNA预测的数据库蛋白序列与UStags进行比对，通过分析UStags及其前缀、后缀序列中存在的差异，推断潜在的序列修饰类型。借助此从头测序-UStags联合方法，我们在多个酵母蛋白序列中发现了由氨基酸突变和/或预测蛋白序列发生多重修饰所导致的意外变异。为确定假阳性率（false discovery rate），我们独立采用两个随机（假）数据库开展序列匹配实验，最终估算该方法的假阳性率≤3%。本研究还对影响该方法可靠性（如存在从头测序噪声残基与冗余序列的情况）与灵敏度的各类因素进行了探究与阐述。该联合从头测序-UStags方法可对已报道的UStags方法进行有效补充，助力新型蛋白质修饰的发现。