Internal amino acid sequence analysis of proteins separated by one- or two-dimensional gel electrophoresis after in situ protease digestion on nitrocellulose.

Abstract

We have developed a general two-step method for obtaining peptide fragments for sequence analysis from picomole quantities of proteins separated by gel electrophoresis. After separation by one-or two-dimensional polyacrylamide gel electrophoresis, proteins are electrophoretically transferred (electroblotted) onto nitrocellulose, the protein-containing regions are detected by reversible staining and are cut out, and each protein is digested in situ by proteolytic enzymes such as trypsin or staphylococcal V-8 protease. The resulting peptide fragments are separated by narrow-bore reverse-phase HPLC, collected, and sequenced in a gas-phase sequenator. Excellent peptide recoveries and the absence of extraneous contaminants in the separation of the peptide fragment mixture allow the generation of extensive internal sequence information from picomole amounts of protein. The method thus overcomes the problem of obtaining amino acid sequence data from Nterminally blocked proteins and provides multiple, independent stretches of sequence that can be used to generate oligonucleotide probes for molecular cloning and/or used to search sequence data bases for related proteins. This method has been successfully applied to the routine amino acid sequence analysis of a wide range of proteins isolated from oneand two-dimensional polyacrylamide gels.Protein sequence analysis is central to modern biological research. Amino acid sequence data can aid in gene isolation either through the use of synthetic oligonucleotides (1) or through the use of antisera of predetermined specificity (2). Sequence data can also be used to correlate the gene structure with the expressed polypeptide chain, to map active sites and domain boundaries, to define posttranslational modifications, or to establish evolutionary relationships between proteins (3).Currently, 20 pmol amounts of protein can give limited N-terminal sequence information using optimized isolation methods (4) and a commercially available gas-phase sequenator (5). It is desirable to develop a method of comparable sensitivity for internal amino acid sequence analysis of proteins for the following reasons:(i) Many proteins are not susceptible to the Edman degradation. This is assumed to be due to artefactual or biosynthetic blocking of the a-amino groups of the proteins. The nature of the modified N terminus is not easily determined and only a few of the modifications can be chemically or enzymatically reversed (6). Therefore, the most general way to obtain sequence information from a protein with a blocked a-amino group is to sequence peptide fragments generated by cleavage of the polypeptide chain.(ii) In the molecular cloning of genes through the use of synthetic oligodeoxynucleotides reverse translated from the protein sequence, it is advantageous to screen appropriate cDNA libraries with either a set of multiple probes or a single probe with minimum degeneracy to reduce the incidence of "false positives." Furthermore, the use of an oligodeoxynucleotide probe derived from the N...

Mass spectrometric identification of dystrophin isoform Dp427 by on-membrane digestion of sarcolemma from skeletal muscle

“…Nitrocellulose strips were then blocked with 0.5% polyvinylpyrrolidone (PVP-40) at 37°C for 37 min with gentle shaking [16]. Membranes were subsequently washed five times with distilled water.…”

“…doi:10.1016/j.ab.2010.05.017 methodology are better protein sequence coverage and a shorter digestion time as compared with in-gel techniques. This reduces complications due to trypsin autolysis and makes this method especially suitable for the mass spectrometric identification of lowabundance and large membrane-associated proteins [16].…”

“…In general, proteins adsorbed onto nitrocellulose sheets are more accessible to proteases, increasing the digestion efficiency [14]. On-membrane trypsination methods have been applied in various biochemical studies [14][15][16][17]. The advantages of on-membrane 0003-2697/$ -see front matter Ó…”

High‐Sensitivity sequencing of large proteins: Partial structure of the rapamycin‐fkbp12 target

“…Since its introduction (Aebersold et al, 1987), in situ digestion of proteins attached to membranes has become a standard tool of protein chemists. Surprisingly, few significant modifications of the original recipe have been suggested, except for the potential use of different enzymes (Tempst et al, 1990;Fernandez et al, 1994) and the inclusion of detergent to promote elution of fragments from PVDF membranes (Fernandez et al, 1992).…”

Analytical and micropreparative peptide mapping by high performance liquid chromatography/electrospray mass spectrometry of proteins purified by gel electrophoresis

“…The most general relevant techniques include chemical or enzymatic fragmentation of isolated polypeptides in the gel matrix following electrophoresis (Eckerskorn & Lottspeich, 1989;Vanfleteren et al, 1992) or, after electrotransfer onto a suitable matrix, fragmentation on the electroblotting support (Aebersold et al, 1987;Iwamatsu, 1992;Patterson et al, 1992). In most cases, cleavage fragments are separated by reverse-phase high performance liquid chromatography, detected by UV absorbance of the peptide bond, and collected for further analyses such as peptide sequencing.…”