Proteomic profiling of erythrocyte proteins by proteolytic digestion chip and identification using two-dimensional electrospray ionization tandem mass spectrometry.
Self-assembled monolayers (SAMs) on coinage metal provide versatile modeling systems for studies of interfacial electron transfer, biological interactions, molecular recognition, and other interfacial phenomena. The bonding of enzyme to SAMs of alkanethiols onto gold surfaces is exploited to produce an enzyme chip. In this work, the attachment of trypsin to a SAMs surface of 11-mercaptoundecanoic acid was achieved using water soluble N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide as coupling agent. A two-dimensional liquid-phase separation scheme coupled with mass spectrometry is presented for proteomic analysis of erythrocyte proteins. The application of proteomics, particularly with reference to analysis of proteins, will be described. Surface analyses have revealed that the X-ray Photoelectron Spectroscopy (XPS) C1s and N1s core levels illustrate the immobilization of trypsin. These data are also in good agreement with Fourier Transformed Infrared Reflection-Attenuated Total Reflection (FTIR-ATR) spectra for the peaks at Amide I and Amide II. Using two-dimensional nano-high performance liquid chromatography electrospray ionization tandem mass spectrometry (2D nano-HPLC-ESI-MS/MS) system observations, analytical results have demonstrated the erythrocyte proteins digestion of the immobilized trypsin on the functionalized SAMs surface. For such surfaces, it also shows the enzyme digestion ability of the immobilized trypsin. The experiment results revealed the identification of 272 proteins from erythrocyte protein sample. The terminal groups of the SAMs structure can be further functionalized with biomolecules or antibodies to develop surface-base diagnostics, biosensors, or biomaterials.
Tyan
Yu-Chang
YC
Department of Environmental & Occupational Health, National Cheng Kung University, No.1, Ta-Hsueh Road, Tainan 701, Taiwan, Republic of China. yctyan@mail.ncku.edu.tw
Jong
Shiang-Bin
SB
Liao
Jiunn-Der
JD
Liao
Pao-Chi
PC
Yang
Ming-Hui
MH
Liu
Chia-Yuan
CY
Klauser
Ruth
R
Himmelhaus
Michael
M
Grunze
Michael
M
D000328:Adult; D004912:Erythrocytes / Q000737:chemistry*; D006801:Humans; D046208:Microchip Analytical Procedures; D010447:Peptide Hydrolases / Q000378:metabolism; D011506:Proteins / Q000032:analysis*; D040901:Proteomics / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization; D017550:Spectroscopy, Fourier Transform Infrared; D014357:Trypsin / Q000378:metabolism
D011506:Proteins; D010447:Peptide Hydrolases; D014357:Trypsin