Enrichment of serum low-molecular-weight proteins using C18 absorbent under urea/dithiothreitol denatured environment.
Serum low-molecular-weight proteins (LMWPs, molecular weight<30kDa) are closely related to the body physiological and pathological situations, whereas many difficulties are encountered when enriching and fractionating them. Using C(18) absorbent (100 A) enrichment and fractionation under urea/dithiothreitol (DTT) denatured environment followed by 60% acetonitrile (ACN) elution, serum LMWPs could be enriched more than 100-fold and were evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional gel electrophoresis (2-DE), and isotope-coded affinity tag (ICAT) labeling quantification. Proteins existing in human serum at low nanograms/milliliter (ng/ml) levels, such as myeloid-related proteins (MRPs), could be identified directly from 2-DE coupled with matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF MS) and LTQ-Orbitrap MS. Sixteen proteins were confidentially identified and quantified using ICAT labeling and liquid chromatography-tandem mass spectrometry (LC-MS/MS). By virtue of its easy operation and high reproducibility to process large quantity complex serum samples, this method has potential uses in enriching LMWPs either in serum or in cell and tissue samples.
Wu
Jing
J
National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871, People's Republic of China.
An
Yuan
Y
Pu
Hai
H
Shan
Yue
Y
Ren
Xiaoqing
X
An
Mingrui
M
Wang
Qingsong
Q
Wei
Shicheng
S
Ji
Jianguo
J
D001798:Blood Proteins / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification*; D002460:Cell Line; D004229:Dithiothreitol / Q000737:chemistry*; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry / Q000378:metabolism; D008970:Molecular Weight; D011489:Protein Denaturation; D015203:Reproducibility of Results; D012117:Resins, Synthetic / Q000737:chemistry*; D012709:Serum Albumin / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014508:Urea / Q000737:chemistry*
D001798:Blood Proteins; D007074:Immunoglobulin G; D012117:Resins, Synthetic; D012709:Serum Albumin; D014508:Urea; D004229:Dithiothreitol