title: Applying multiple proteases to direct digestion of hundred-scale cell samples for proteome analysis.
abstract: BACKGROUND
  Analyzing the proteome on the scale of only several hundred cells with mass spectrometry has great significance for applications with limited sample amounts. We applied multiple proteases to the direct digestion of cells and compared the identified proteins both qualitatively and quantitatively.
  
  
  METHODS
  Three hundred cells were directly digested by trypsin, chymotrypsin, or the combination of trypsin and chymotrypsin. The peptides were identified using a LTQ-Orbitrap XL, and data were analyzed using MaxQuant software.
  
  
  RESULTS
  Different proteases produced different identified protein numbers. Trypsin proved to be the best choice for generating the largest protein number, while other proteases complemented the identification results of trypsin by increasing protein sequence coverage. Concerning the quantitative perspective, using trypsin would produce the biggest number of proteins quantifiable by intensity-based absolute quantification (iBAQ).
  
  
  CONCLUSIONS
  When hundred-scale cell samples are analyzed, an optimum choice of proteases should be made to realize different analytical objectives.
authors.affiliation: Department of Chemistry, Fudan University, No. 220 Handan Rd., Shanghai, 200433, China.
authors.forename: Qi
authors.initials: Q
authors.lastname: Chen
authors.affiliation: Department of Chemistry, Fudan University, No. 220 Handan Rd., Shanghai, 200433, China.
authors.forename: Guoquan
authors.initials: G
authors.lastname: Yan
authors.affiliation: Department of Chemistry, Fudan University, No. 220 Handan Rd., Shanghai, 200433, China.
authors.forename: Xiangmin
authors.initials: X
authors.lastname: Zhang
mesh_terms: D000595:Amino Acid Sequence; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin / Q000378:metabolism*; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D010449:Peptide Mapping / Q000379:methods*; D020543:Proteome / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics; D014357:Trypsin / Q000378:metabolism*
chemical_list: D010446:Peptide Fragments; D020543:Proteome; D002918:Chymotrypsin; D014357:Trypsin