Facilitating the hyphenation of CIEF and MALDI-MS for two-dimensional separation of proteins.
Both CIEF and MALDI-MS are frequently used in protein analysis, but hyphenation of the two has not been investigated proportionally. One of the major reasons is that the additives (such as carrier ampholytes and detergent) in CIEF severely suppress the MALDI-MS signal, which hampers the hyphenation of the two. In this paper, we develop a simple means to alleviate the above signal-suppressing effect. We first deposit 1 microL of water onto a MALDI-MS target, deliver a fraction of CIEF-separated protein (approximately 0.1 microL) to the water droplet, evaporate the solvent, add 0.5 microL of MALDI matrix to the sample spot, dry the matrix and move the target plate to a MALDI-TOF-MS for mass spectrum measurement. We optimize the droplet volume and the laser-ablation region. Under the optimized conditions, we improve the S/N by two- to tenfold. We also apply this method for 2-D separations of standard proteins and apolipoprotein A-I, a membrane protein expressed in Escherichia coli cells.
Cheng
Chang
C
Department of Chemistry and Biochemistry, University of Oklahoma, Norman, OK, USA.
Lu
Joann J
JJ
Wang
Xiayan
X
Roberts
Jonathan
J
Liu
Shaorong
S
D000818:Animals; D016632:Apolipoprotein A-I / Q000235:genetics / Q000302:isolation & purification; D019075:Electrophoresis, Capillary / Q000295:instrumentation / Q000379:methods*; D004867:Equipment Design; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D007525:Isoelectric Focusing / Q000295:instrumentation / Q000379:methods; D011506:Proteins / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D016632:Apolipoprotein A-I; D011506:Proteins