Terry Doris E, Umstot Edward, Desiderio Dominic M
The Charles B. Stout Neuroscience Mass Spectrometry Laboratory, The University of Tennessee Center for Health Science, Memphis, 38163, USA.
J Am Soc Mass Spectrom. 2004 Jun;15(6):784-94. doi: 10.1016/j.jasms.2004.02.005.
Proteomics requires an optimized level of sample-processing, including a minimal sample-processing time and an optimal peptide recovery from protein digests, in order to maximize the percentage sequence coverage and to improve the accuracy of protein identification. The conventional methods of protein characterization from one-dimensional or two-dimensional gels include the destaining of an excised gel piece, followed by an overnight in-gel enzyme digestion. The aims of this study were to determine whether: (1) stained gels can be used without any destaining for trypsin digestion and mass spectrometry (MS); (2) tryptic peptides can be recovered from a matrix-assisted laser desorption/ionization (MALDI) target plate for a subsequent analysis with liquid chromatography (LC) coupled to an electrospray ionization (ESI) quadrupole ion trap MS; and (3) an overnight in-gel digestion is necessary for protein characterization with MS. These three strategies would significantly improve sample throughput. Cerebrospinal fluid (CSF) was the model biological fluid used to develop these methods. CSF was desalted by gel filtration, and CSF proteins were separated by two-dimensional gel electrophoresis (2DGE). Proteins were visualized with either silver, Coomassie, or Stains-All (counterstained with silver). None of the gels was destained. Protein spots were in-gel trypsin digested, the tryptic peptides were purified with ZipTip, and the peptides were analyzed with MALDI and ESI MS. Some of the samples that were spotted onto a wax-coated MALDI target plate were recovered and analyzed with ESI MS. All three types of stained gels were compatible with MALDI and ESI MS without any destaining. In-gel trypsin digestion can be performed in only 10-60 min for protein characterization with MS, the sample can be recovered from the MALDI target plate for use in ESI MS, and there was a 90% reduction in sample-processing time from overnight to ca. 3 h.
蛋白质组学需要优化的样品处理水平，包括最短的样品处理时间和从蛋白质消化物中获得最佳的肽回收率，以便最大化序列覆盖率百分比并提高蛋白质鉴定的准确性。从一维或二维凝胶进行蛋白质表征的传统方法包括对切下的凝胶块进行脱色，然后进行过夜的胶内酶消化。本研究的目的是确定：（1）染色后的凝胶是否可以不经任何脱色用于胰蛋白酶消化和质谱分析（MS）；（2）胰蛋白酶肽是否可以从基质辅助激光解吸/电离（MALDI）靶板上回收，用于随后与电喷雾电离（ESI）四极杆离子阱质谱联用的液相色谱（LC）分析；以及（3）用MS进行蛋白质表征是否需要过夜胶内消化。这三种策略将显著提高样品通量。脑脊液（CSF）是用于开发这些方法的模型生物流体。通过凝胶过滤对CSF进行脱盐，并通过二维凝胶电泳（2DGE）分离CSF蛋白质。用银染、考马斯亮蓝或全染剂（用银复染）对蛋白质进行可视化。所有凝胶均未脱色。对蛋白质斑点进行胶内胰蛋白酶消化，用ZipTip纯化胰蛋白酶肽，并用MALDI和ESI MS分析这些肽。一些点样到涂蜡的MALDI靶板上的样品被回收并用ESI MS分析。所有三种类型的染色凝胶在不经任何脱色的情况下都与MALDI和ESI MS兼容。对于用MS进行蛋白质表征，胶内胰蛋白酶消化仅需10 - 60分钟即可完成，样品可从MALDI靶板上回收用于ESI MS，并且样品处理时间从过夜减少了90%，降至约3小时。
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