Visualization of proteins by modification of lysines, cysteines, and phosphorylated serines facilitates sample preparation for microsequencing

被引:9
作者
Hsi, KL [1 ]
O'Neill, SA [1 ]
Dupont, DR [1 ]
Yuan, PM [1 ]
机构
[1] PE Appl Biosyst, Foster City, CA 94404 USA
关键词
D O I
10.1006/abio.1998.2582
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
A procedure for visualization and sensitive detection of protein during sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequent sample preparation for sequence analysis is described. This procedure utilizes either fluorescent, or visible tags for certain amino acids in protein molecules, e.g., lysines modified with dansyl/dabsyl chloride and cystines/cysteines or phosphorylated serines modified with iodoacetamidofluorescein (I-15) after proper sample pretreatments. Modifications are performed prior to SDS-PAGE, eliminating the need for fixing, staining, and destaining as required for the conventional procedures. After electrophoresis, the fluorescent or visible bands are excised from the gel, homogenized in microcentrifuge tubes, and soaked in an appropriate buffer to release the separated proteins into solution, Enzymatic digestion can then be carried out in solution for better efficiency of digestion and recovery. The subsequent HPLC mapping and collection of protein digests are performed on PE Applied Biosystems Model 173A MicroBlotter. The separated peptides containing tagged amino acids are visible on the PVDF membrane and can be excised for direct sequence analysis. This approach has been employed for selectively isolating the lysine, cysteine, or phosphorylated serine containing peptides using model proteins. The sequencing results of the peptides generated from premodified proteins demonstrate that this approach facilitates sample preparation for microsequence analysis at low picomole level, Overall. recoveries of 20-30% by sequencing initial yields have been achieved using our model proteins. (C) 1998 Academic Press.
引用
收藏
页码:38 / 47
页数:10
相关论文
共 13 条
[1]  
CLARK RC, 1967, INT J BIOCHEM, V11, P577
[2]   SIGNAL INTEGRATION AT THE LEVEL OF PROTEIN-KINASES, PROTEIN PHOSPHATASES AND THEIR SUBSTRATES [J].
COHEN, P .
TRENDS IN BIOCHEMICAL SCIENCES, 1992, 17 (10) :408-413
[3]   QUANTITATIVE AND SELECTIVE FLUOROPHORE LABELING OF PHOSPHOSERINE ON PEPTIDES AND PROTEINS - CHARACTERIZATION AT THE ATTOMOLE LEVEL BY CAPILLARY ELECTROPHORESIS AND LASER-INDUCED FLUORESCENCE [J].
FADDEN, P ;
HAYSTEAD, TAJ .
ANALYTICAL BIOCHEMISTRY, 1995, 225 (01) :81-88
[5]   A strategy to obtain internal sequence information from blotted proteins after initial N-terminal sequencing [J].
Hsi, KL ;
Werner, WE ;
Zieske, LR ;
Grimley, CH ;
ONeill, SA ;
Kochersperger, ML ;
Yamada, K ;
Yuan, PM .
TECHNIQUES IN PROTEIN CHEMISTRY VIII, 1997, 8 :91-98
[6]  
Hsi KL, 1997, PROTEIN PEPTIDE LETT, V4, P1
[7]   ON TARGET WITH A NEW MECHANISM FOR THE REGULATION OF PROTEIN-PHOSPHORYLATION [J].
HUBBARD, MJ ;
COHEN, P .
TRENDS IN BIOCHEMICAL SCIENCES, 1993, 18 (05) :172-177
[8]   INTERNAL SEQUENCES FROM PROTEINS DIGESTED IN POLYACRYLAMIDE GELS [J].
JENO, P ;
MINI, T ;
MOES, S ;
HINTERMANN, E ;
HORST, M .
ANALYTICAL BIOCHEMISTRY, 1995, 224 (01) :75-82
[9]  
KOCHERSPERGER LM, 1994, PROTEIN SCI S1, V3, P98
[10]  
LUNDBLAD RL, 1991, CHEM REAGENTS PROTEI, P59