Mateos Jesús, Pintor-Iglesias Alejandra, Fernández-Puente Patricia, García-Camba Marta, Ruiz-Romero Cristina, Doménech Nieves, Blanco Francisco J
Rheumatology Division, Proteomics Unit-ProteoRed/ISCIII, INIBIC-Hospital Universitario A Coruña , As Xubias 84, 15006 A Coruña, Spain.
J Proteome Res. 2014 Apr 4;13(4):1930-7. doi: 10.1021/pr401046u. Epub 2014 Mar 3.
We tested a semiautomated protocol for the proper storage and conservation in a hospital biobank of tryptic peptide extracts coming from samples with low and high protein complexity for subsequent mass spectrometry analysis. Low-complexity samples (serum albumin, serotransferrin. and alpha-S1-casein) were loaded in replicates in SDS-PAGE and subjected to standard in-gel trypsin digestion. For LC-MALDI-TOF/TOF analysis, purified β-galactosidase and human serum samples were in-solution digested following standard procedures and desalted with C18 stage-tips. In both cases, peptides extracts were aliquoted in individually 2D coded tubes, vacuum-dried, barcode-read, and stored in an automated -20 °C freezer in the Biobank facility. Samples were kept dried at -20 °C until the corresponding time-point of analysis, then reconstituted in the proper buffer and analyzed by either MALDI-TOF/TOF (peptide fingerprinting and MS/MS) or LC-MALDI-TOF/TOF following a highly reproducible pattern to ensure the reproducibility of the results. Protein identification was done with either Mascot or Protein Pilot as search engines using constant parameters. Over a period of 1 year we checked six different time points at days 0, 7, 30, 90, 180, and 365. We compared MS and MS/MS protein score, number of identified peptides, and coverage of the identified proteins. In the low complexity samples, the number of peptides detected gradually decreased over time, especially affecting the MS score. However, two of the three proteins - serum albumin and serotransferrin - were identified by both PMF and MS/MS at day 90. By day 180, only MS/MS identification in some replicates was possible. By LC-MS/MS, β-galactosidase and the most abundant serum proteins were identified with good scores at all time points even by day 365, with no detectable peptide loss or decrease in the fragmentation efficiency, although a progressive decrease in peptide intensity indicates that detection of low abundant proteins could not be optimal after very long periods of time. Our results encourage us to use the biobank facility in the future for long-term storage - up to 3 months - of dried peptide extracts.
我们测试了一种半自动方案，用于在医院生物样本库中妥善储存和保存来自低蛋白复杂性和高蛋白复杂性样本的胰蛋白酶肽提取物，以供后续质谱分析使用。低复杂性样本（血清白蛋白、转铁蛋白和α-S1-酪蛋白）一式多份加载到SDS-PAGE中，并进行标准的胶内胰蛋白酶消化。对于LC-MALDI-TOF/TOF分析，纯化的β-半乳糖苷酶和人血清样本按照标准程序进行溶液内消化，并用C18阶段尖端进行脱盐。在这两种情况下，肽提取物均分装到单独的二维编码管中，真空干燥，读取条形码，并储存在生物样本库设施中-20°C的自动冷冻柜中。样本在-20°C下保持干燥，直至相应的分析时间点，然后用适当的缓冲液复溶，并按照高度可重复的模式通过MALDI-TOF/TOF（肽指纹图谱和MS/MS）或LC-MALDI-TOF/TOF进行分析，以确保结果的可重复性。使用Mascot或Protein Pilot作为搜索引擎，采用恒定参数进行蛋白质鉴定。在1年的时间里，我们在第0、7、30、90、180和365天检查了六个不同的时间点。我们比较了MS和MS/MS蛋白质得分、鉴定出的肽数量以及鉴定出的蛋白质的覆盖率。在低复杂性样本中，随着时间的推移，检测到的肽数量逐渐减少，尤其影响MS得分。然而，三种蛋白质中的两种——血清白蛋白和转铁蛋白——在第90天通过PMF和MS/MS均被鉴定出来。到第180天，仅在一些重复样本中可以进行MS/MS鉴定。通过LC-MS/MS，即使到第365天，β-半乳糖苷酶和最丰富的血清蛋白在所有时间点均以良好的得分被鉴定出来，没有可检测到的肽损失或碎片化效率降低，尽管肽强度逐渐降低表明经过很长时间后低丰度蛋白质的检测可能不是最佳的。我们的结果鼓励我们未来在生物样本库设施中对干燥的肽提取物进行长达3个月的长期储存。
