Department of Chemical Physiology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
J Proteome Res. 2013 May 3;12(5):2177-84. doi: 10.1021/pr400027m. Epub 2013 Apr 9.
A modified multidimensional protein identification technology (MudPIT) separation was coupled to an LTQ Orbitrap Velos mass spectrometer and used to rapidly identify the near-complete yeast proteome from a whole cell tryptic digest. This modified online two-dimensional liquid chromatography separation consists of 39 strong cation exchange steps followed by a short 18.5 min reversed-phase (RP) gradient. A total of 4269 protein identifications were made from 4189 distinguishable protein families from yeast during log phase growth. The "Micro" MudPIT separation performed as well as a standard MudPIT separation in 40% less gradient time. The majority of the yeast proteome can now be routinely covered in less than a days' time with high reproducibility and sensitivity. The newly devised separation method was used to detect changes in protein expression during cellular quiescence in yeast. An enrichment in the GO annotations "oxidation reduction", "catabolic processing" and "cellular response to oxidative stress" was seen in the quiescent cellular fraction, consistent with their long-lived stress resistant phenotypes. Heterogeneity was observed in the stationary phase fraction with a less dense cell population showing reductions in KEGG pathway categories of "Ribosome" and "Proteasome", further defining the complex nature of yeast populations present during stationary phase growth. In total, 4488 distinguishable protein families were identified in all cellular conditions tested.
改良多维蛋白质鉴定技术（MudPIT）分离与 LTQ Orbitrap Velos 质谱仪相连，用于快速鉴定整个细胞胰蛋白酶消化物中的近完整酵母蛋白质组。这种改良的在线二维液相色谱分离由 39 个强阳离子交换步骤组成，随后是 18.5 分钟的短反相（RP）梯度。在对数生长期，从酵母的 4189 个可区分蛋白家族中鉴定出了 4269 种蛋白。“微型”MudPIT 分离在 40%的梯度时间内与标准 MudPIT 分离效果一样好。现在，大多数酵母蛋白质组可以在不到一天的时间内，以高重复性和灵敏度进行常规覆盖。新设计的分离方法用于检测酵母细胞静止期蛋白表达的变化。在细胞静止期的细胞部分观察到 GO 注释“氧化还原”、“分解代谢加工”和“细胞对氧化应激的反应”的富集，与它们的长寿命应激抗性表型一致。在静止期细胞部分观察到异质性，细胞密度较低的细胞群中“核糖体”和“蛋白酶体”的 KEGG 途径分类减少，进一步定义了静止期生长过程中存在的酵母群体的复杂性质。总共在所有测试的细胞条件下鉴定出了 4488 个可区分蛋白家族。
