Alternative Ion-Pairing Modifiers Should Be Investigated in Low-Input and Single-Cell Proteomics

A recent study demonstrated a substantial increase in the 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. Given the strictly limited material and counterintuitive observations by others in the emerging field of single-cell proteomics, we chose to investigate this alternative modifier in the analysis of subnanogram proteome dilutions. When digest standards as low as 20 pg total load on the column were evaluated, AA led to increased proteome coverage at every peptide load assessed. Relative improvements were more apparent at lower concentrations, with a 20 pg peptide digest demonstrating a striking 1.8-fold increase to over 2000 protein groups identified in a 30 min analysis. Furthermore, we find that this increase in signal can be leveraged to reduce ramp times, leading to 1.7× more scans across each peak and improvements in quantification, as measured by %CVs. These results can be reproduced on multiple trapped ion mobility instruments. When evaluating single cancer cells, approximately 13% more peptide groups were identified on average when employing AA in the place of FA. These results suggest that ion pairing modifiers and other additives warrant re-evaluation in the context of low-input and single-cell proteomics. All vendor raw and processed data are available through ProteomeXchange as PXD046002 and PXD051590.

近期一项研究表明，在鸟枪法蛋白质组学（shotgun proteomics）中，采用0.5%乙酸（AA）作为离子对改性剂替代传统使用的0.1%甲酸（FA）时，肽信号强度与对应蛋白质组覆盖度均得到显著提升。鉴于单细胞蛋白质组学这一新兴领域存在样本量极为有限的痛点，且已有其他研究者得出与直觉相悖的观测结果，我们选择对这一替代改性剂展开研究，用于亚纳克级蛋白质组稀释液的分析。当评估柱上总上样量低至20皮克的酶解标准品时，在所有测试的肽上样量条件下，乙酸均能提升蛋白质组覆盖度。低浓度下的相对提升效果更为显著：在30分钟的分析时长内，20皮克肽酶解样品的鉴定蛋白质群组数量增幅可达惊人的1.8倍，突破2000个。此外，我们发现可利用信号强度的提升来缩短梯度升温时间，此举可使每个色谱峰的扫描次数增加1.7倍，并通过变异系数百分比（%CVs）量化得出定量性能的改善。上述结果可在多台捕集离子淌度仪器上重复实现。在对单个癌细胞进行评估时，采用乙酸替代甲酸的策略平均可多鉴定出约13%的肽段群组。上述结果表明，在低投入量与单细胞蛋白质组学的研究场景中，离子对改性剂与其他添加剂有必要被重新评估。所有供应商提供的原始数据与处理后数据均可通过蛋白质组交换数据库（ProteomeXchange）获取，编号为PXD046002与PXD051590。