Optimization of protein identifications through the use of different chromatographic approaches and bioinformatic pipelines.

IF 1.8 3区化学 Q4 BIOCHEMICAL RESEARCH METHODS Rapid Communications in Mass Spectrometry Pub Date : 2025-01-15 DOI:10.1002/rcm.9937

Jesus D Castaño, Francis Beaudry

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引用次数: 0

Abstract

Rationale: Selection of proteomic workflows for a given project can be a daunting task. This research provides a guide outlining the impact on protein identification of different steps such as chromatographic separation, data acquisition strategies, and bioinformatic pipelines. The data presented here will help experts and nonexpert proteomic users to increase proteome coverage and peptide identification.

Methods: HeLa protein digests were analyzed through different C18 chromatographic columns (15 and 50 cm in length), using top 12 data-dependent acquisition (DDA), top 20 DDA, and data-independent acquisition (DIA) with a nanospray source in positive mode in a Thermo Q Exactive instrument. The raw data were analyzed using different search engines, rescoring approaches, and multi-engine searches. The results were analyzed in the context of peptide and protein identifications, precursor properties, and computation requirements to understand the differences between methods.

Results: Our results showed that higher column lengths and top N DDA approaches were able to significantly increase protein identifications. The use of multiple search engines yielded limited gains, whereas the use of rescoring methods clearly outperformed other strategies. Finally, DIA approaches, although successful at generating new identifications, had a limited performance influenced by the previous collection of DDA data, which could prohibitively increase instrument time. Nonetheless, the use of library-free methods showed promising results.

Conclusions: Our results highlight the impact of different experimental approaches on proteome coverage. Changes in chromatographic columns, data acquisition, or bioinformatic analysis can significantly increase the number of protein identifications (>400%). Thus, this research provides a reference upon which to build a successful proteomic workflow with different considerations at every step.

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通过使用不同的色谱方法和生物信息学管道优化蛋白质鉴定。

理由：为特定项目选择蛋白质组工作流程是一项艰巨的任务。本研究提供了一份指南，概述了色谱分离、数据采集策略和生物信息管道等不同步骤对蛋白质鉴定的影响。方法：通过不同的 C18 色谱柱（长度分别为 15 厘米和 50 厘米），在 Thermo Q Exactive 仪器的正向模式下使用前 12 位数据依赖性采集（DDA）、前 20 位数据依赖性采集（DDA）和纳米喷雾源数据无关性采集（DIA），对 HeLa 蛋白消化液进行分析。使用不同的搜索引擎、重新评分方法和多引擎搜索对原始数据进行了分析。我们结合肽和蛋白质鉴定、前体特性和计算要求对结果进行了分析，以了解不同方法之间的差异：结果：我们的研究结果表明，较高的柱长和前 N DDA 方法能够显著提高蛋白质鉴定率。使用多个搜索引擎的收益有限，而使用重评分方法的效果明显优于其他策略。最后，DIA 方法虽然能成功地产生新的鉴定结果，但其性能受到之前 DDA 数据收集的影响，可能会过多地增加仪器时间。尽管如此，无库方法的使用还是取得了可喜的成果：我们的研究结果凸显了不同实验方法对蛋白质组覆盖率的影响。色谱柱、数据采集或生物信息分析的改变可显著增加蛋白质鉴定的数量（>400%）。因此，这项研究为建立成功的蛋白质组工作流程提供了参考，每一步都有不同的考虑因素。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Rapid Communications in Mass Spectrometry 化学-分析化学

CiteScore

4.10

自引率

5.00%

发文量

219

审稿时长

2.6 months

期刊介绍： Rapid Communications in Mass Spectrometry is a journal whose aim is the rapid publication of original research results and ideas on all aspects of the science of gas-phase ions; it covers all the associated scientific disciplines. There is no formal limit on paper length ("rapid" is not synonymous with "brief"), but papers should be of a length that is commensurate with the importance and complexity of the results being reported. Contributions may be theoretical or practical in nature; they may deal with methods, techniques and applications, or with the interpretation of results; they may cover any area in science that depends directly on measurements made upon gaseous ions or that is associated with such measurements.