Eberhard Colten D, Orsburn Benjamin C
The Department of Pharmacology and Molecular Sciences.
The Johns Hopkins University School of Medicine, Baltimore, MD, USA, 21205.
bioRxiv. 2024 Jun 10:2023.08.01.551522. doi: 10.1101/2023.08.01.551522.
A recent study demonstrated a substantial increase in peptide signal and corresponding proteome coverage when employing 0.5% acetic acid (AA) as the ion pairing modifier in place of the 0.1% formic acid traditionally used in shotgun proteomics. In this study, we investigated the effect of modifier in the context of sub-nanogram and single cell proteomics (SCP). We first evaluated a tryptic digest standard down to 20 picograms total load on column on a TIMSTOF SCP system. In line with the previous results, we observed a signal increase when using AA, leading to increased proteome coverage at every peptide load assessed. Relative improvements were more apparent at lower concentrations, with a 20 picogram peptide digest demonstrating a striking 1.8-fold increase to over 2,000 protein groups identified in a 30 minute analysis. Furthermore, we find that this increase in signal can be leveraged to reduce ramp times, leading to 1.7x more scans across each peak and improvements in quantification as measured by %CVs. When evaluating single cancer cells, approximately 13% more peptide groups were identified on average when employing AA in the place of FA. All vendor raw and processed data are available through ProteomeXchange as PXD046002 and PXD051590.
最近的一项研究表明，在鸟枪法蛋白质组学中，用0.5%的乙酸（AA）作为离子对修饰剂取代传统使用的0.1%甲酸时，肽信号和相应的蛋白质组覆盖范围大幅增加。在本研究中，我们在亚纳克和单细胞蛋白质组学（SCP）背景下研究了修饰剂的作用。我们首先在TIMSTOF SCP系统上评估了低至20皮克总上样量的胰蛋白酶消化标准品。与之前的结果一致，我们观察到使用AA时信号增强，在评估的每个肽上样量下蛋白质组覆盖范围都有所增加。在较低浓度下相对改善更为明显，20皮克的肽消化物在30分钟分析中鉴定出的蛋白质组超过2000个，显著增加了1.8倍。此外，我们发现这种信号增强可用于减少扫描时间，每个峰的扫描次数增加1.7倍，并提高了以%CVs衡量的定量准确性。在评估单个癌细胞时，使用AA代替FA时平均鉴定出的肽组数量多出约13%。所有供应商的原始数据和处理后的数据可通过ProteomeXchange获得，编号分别为PXD046002和PXD051590。
