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No single perfect protocol can be used to comprehensively characterize the proteome of membrane fraction. Here, we used cow milk fat globule membrane (MFGM) proteome analysis to assess six sample preparation procedures including one in\u2010gel and five in\u2010solution digestion approaches prior to LC\u2010MS\/MS analysis. The largest number of MFGM proteins were identified by suspension trapping (S\u2010Trap) and filter\u2010aided sample preparation (FASP) methods, followed by acetone precipitation without clean\u2010up of tryptic peptides method. Protein identifications with highest average coverage was achieved by Chloroform\/MeOH, in\u2010gel and S\u2010Trap methods. Most distinct proteins were identified by FASP method, followed by S\u2010Trap. Analyses by Venn diagram, principal\u2010component analysis, hierarchical clustering and the abundance ranking of quantitative proteins highlight differences in the MFGM fraction by the all sample preparation procedures. These results reveal the biased proteins\/peptides loss occurred in each protocol. In this study, we found several novel proteins that were not observed previously by in\u2010depth proteomics characterization of MFGM fraction in milk. Thus, a combination of multiple procedures with orthologous properties of sample preparation was demonstrated to improve the protein sequence coverage and expression level accuracy of membrane samples.<\/jats:p>","DOI":"10.1002\/elps.201800042","type":"journal-article","created":{"date-parts":[[2018,4,12]],"date-time":"2018-04-12T03:48:34Z","timestamp":1523504914000},"page":"2332-2339","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":72,"title":["Evaluation of six sample preparation procedures for qualitative and quantitative proteomics analysis of milk fat globule membrane"],"prefix":"10.1002","volume":"39","author":[{"given":"Yongxin","family":"Yang","sequence":"first","affiliation":[{"name":"Institute of Biotechnology Cornell University  Ithaca NY USA"},{"name":"Institute of Animal Science and Veterinary Medicine Anhui Academy of Agricultural Sciences  Hefei P. R. 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