Spahr C S, Davis M T, McGinley M D, Robinson J H, Bures E J, Beierle J, Mort J, Courchesne P L, Chen K, Wahl R C, Yu W, Luethy R, Patterson S D
Departments of Biochemistry and Genetics, Amgen, Thousand Oaks, CA, USA.
Proteomics. 2001 Jan;1(1):93-107. doi: 10.1002/1615-9861(200101)1:1<93::AID-PROT93>3.0.CO;2-3.
The proteome of normal male urine from a commercial pooled source has been examined using direct liquid chromatography-tandem mass spectrometry (LC-MS/MS). The entire urinary protein mixture was denatured, reduced and enzymatically digested prior to LC-MS/MS analysis using a hybrid-quadrupole time-of-flight mass spectrometer (Q-TOF) to perform data-dependent ion selection and fragmentation. To fragment as many peptides as possible, the mixture was analyzed four separate times, with the mass spectrometer selecting ions for fragmentation from a subset of the entire mass range for each run. This approach requires only an autosampler on the HPLC for automation (i.e, unattended operation). Across these four analyses, 1.450 peptide MS/MS spectra were matched to 751 sequences to identify 124 gene products (proteins and translations of expressed sequence tags). Interestingly, the experimental time for these analyses was less than that required to run a single two-dimensional gel.
利用直接液相色谱-串联质谱法（LC-MS/MS）对来自商业混合来源的正常男性尿液蛋白质组进行了检测。在使用混合四极杆飞行时间质谱仪（Q-TOF）进行数据依赖型离子选择和碎片化处理以进行LC-MS/MS分析之前，将整个尿液蛋白质混合物进行变性、还原和酶解。为了使尽可能多的肽段发生碎片化，该混合物被分四次单独分析，每次运行时质谱仪从整个质量范围内的一个子集中选择离子进行碎片化。这种方法仅需要高效液相色谱仪上的一个自动进样器来实现自动化（即无人值守操作）。在这四次分析中，1450个肽段的MS/MS谱图与751个序列相匹配，从而鉴定出124种基因产物（蛋白质和表达序列标签的翻译产物）。有趣的是，这些分析所需的实验时间比进行一次二维凝胶电泳所需的时间要少。
