Low-molecular-weight human serum proteome using ultrafiltration, isoelectric focusing, and mass spectrometry.
Identification of the serum proteome is a daunting analytical task due to the complex nature of the sample which has an extremely large dynamic range of protein components. This report addresses this issue by using centrifugal ultrafiltration to enrich the low-molecular-weight (LMW) serum proteome while decreasing the amount of abundant high-molecular-weight proteins. Reduction of the complex nature of the sample was achieved by fractionation of the LMW serum proteins using solution-phase isoelectric focusing (IEF). Multiple enzyme digestions are performed and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Analysis of the tandem mass spectra resulted in the identification of 262 proteins belonging to LMW serum proteome. Our results demonstrate the effectiveness of this methodology to isolate and identify LMW proteins with improved confidence in the MS data acquired. In addition, our methodology can be combined with other multidimensional chromatography techniques performed on the peptide level to increase the number of identified proteins.
Harper
Robert G
RG
Protea Biosciences Inc., West Virginia University, Department of Chemistry, Morgantown, USA.
Workman
Sarah R
SR
Schuetzner
Shayne
S
Timperman
Aaron T
AT
Sutton
Jennifer N
JN
D002918:Chymotrypsin / Q000378:metabolism; D004586:Electrophoresis; D006801:Humans; D007525:Isoelectric Focusing / Q000379:methods*; D020543:Proteome; D044967:Serum / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D014357:Trypsin / Q000378:metabolism
D020543:Proteome; D002918:Chymotrypsin; D014357:Trypsin