A desalting step using reversed phase chromatography is a common practice prior to mass spectrometry analysis of proteolytic digests in spite of the detrimental exclusion of the hydrophilic peptides. The detection of such peptides is also important for the complete coverage of protein sequences and the analysis of posttranslational modifications as inquired by regulatory agencies for the commercialization of biotechnological products. The procedure described here, named in-solution buffer-free digestion, simplifies the sample processing and circumvents the above-mentioned limitations by allowing the detection of tryptic hydrophilic peptides via direct ESI-MS analysis. Two DNA recombinant proteins such as HBcAg (hepatitis B core antigen) and fusion VEGF (vascular endothelial growth factor) were analyzed with the proposed in-solution buffer-free digestion allowing the detection of extremely hydrophilic di-, tri- and tetra-peptides, C-terminal His-tail peptide, as well as disulfide-containing peptides. All these molecular species are hardly seen in mass spectrometric analysis using a standard digestion that includes a C18-desalting step. The procedure was also successfully tried on hydrophilic tetra- and hexa-peptides of Ribonuclease B carrying an N-glycosylation site occupied with “high-mannose” N-glycan chains. The in-solution buffer-free digestion constitutes a simple and straightforward approach to analyse the hydrophilic proteolytic peptides which are commonly elusive to the detection by conventional mass spectrometric analysis.

中文翻译：

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通过溶液中无缓冲液胰蛋白酶消化，通过 MS 靶向重组蛋白的亲水区域

尽管会有害地排除亲水性肽，但在对蛋白水解消化物进行质谱分析之前，使用反相色谱法进行脱盐步骤是一种常见做法。此类肽的检测对于蛋白质序列的完整覆盖和翻译后修饰的分析也很重要，正如监管机构要求的生物技术产品商业化。此处描述的程序称为无溶液缓冲液消化，通过直接 ESI-MS 分析允许检测胰蛋白酶亲水肽，从而简化了样品处理并规避了上述限制。两种 DNA 重组蛋白，如 HBcAg（乙型肝炎核心抗原）和融合 VEGF（血管内皮生长因子），使用建议的溶液内无缓冲液消化进行分析，允许检测极亲水的二肽、三肽和四肽， C-末端His-tail肽，以及含二硫键的肽。在使用包括 C18 脱盐步骤的标准消化的质谱分析中几乎看不到所有这些分子种类。该程序还成功地用于核糖核酸酶 B 的亲水性四肽和六肽，其带有一个被“高甘露糖”N-聚糖链占据的 N-糖基化位点。