Identification of 2-D Gel Proteins at the Femtomole Level by Molecular Mass Searching of Peptide Fragments in a Protein Sequence Database

Henzel, William J.; Billeci, Todd M.; Stults, John T.; Wong, Susan; Grimley, Christopher; Watanabe, Colin

doi:10.1016/b978-0-12-194710-1.50006-0

Cited by 5 publications

(2 citation statements)

References 15 publications

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“…The simplest approach is premised on the belief that the protein with the most matches to the measured peptide molecular weights is the correct answer. This approach was used by Yates et al [28], Mann et al [29], and Henzel et al [32]. No scoring is used beyond how many of the peptide masses match to the protein.…”

Section: Protein Identification Using Peptide Mass Mapsmentioning

confidence: 99%

Database searching using mass spectrometry data

1998

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Large-scale DNA sequencing is creating a sequence infrastructure of great benefit to protein biochemistry. Concurrent with the application of large-scale DNA sequencing to whole genome analysis, mass spectrometry has attained the capability to rapidly, and with remarkable sensitivity, determine weights and amino acid sequences of peptides. Computer algorithms have been developed to use the two different types of data generated by mass spectrometers to search sequence databases. When a protein is digested with a site-specific protease, the molecular weights of the resulting collection of peptides, the mass map or fingerprint, can be determined using mass spectrometry. The molecular weights of the set of peptides derived from the digestion of a protein can then be used to identify the protein. Several different approaches have been developed. Protein identification using peptide mass mapping is an effective technique when studying organisms with completed genomes. A second method is based on the use of data created by tandem mass spectrometers. Tandem mass spectra contain highly specific information in the fragmentation pattern as well as sequence information. This information has been used to search databases of translated protein sequences as well as nucleotide databases such as expressed sequence tag (EST) sequences. The ability to search nucleotide databases is an advantage when analyzing data obtained from organisms whose genomes are not yet completed, but a large amount of expressed gene sequence is available (e.g., human and mouse). Furthermore, a strength of using tandem mass spectra to search databases is the ability to identify proteins present in fairly complex mixtures.

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Section: Protein Identification Using Peptide Mass Mapsmentioning

confidence: 99%

Database searching using mass spectrometry data

1998

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“…In cases where the protease specificity is determined solely by the P1 or P′1 residue (Table 11.1.3), the number of cleavages depends on the distribution of these particular amino acids. It is estimated that lysine (Lys), arginine (Arg), glutamic acid (Glu), and aspartic acid (Asp) each account for about 5% of all amino acids in the protein databases (Henzel et al, 1994). In contrast, tryptophan (Trp) is less abundant and glycine (Gly) more abundant.…”

Section: Background Informationmentioning

confidence: 99%

Enzymatic Digestion of Proteins in Solution

Riviere¹,

Tempst

1995

CP Protein Science

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Analysis of protein covalent structure is less complex and more accurate when performed on peptides derived from the larger protein. In contrast to acid-promoted total hydrolysis, peptides are typically generated by selective proteolysis, i.e., by specifically cleaving peptide bonds with endoproteases that have varying degrees of specificity. This unit presents a protocol that can be used to generate peptide fragments from intact, undenatured proteins. Fragments can be analyzed directly by mass spectrometry (MS) or, more often, are first separated by reversed-phase HPLC (RP-HPLC) and then analyzed by MS or automated sequencing. Most proteins are resistant to enzymatic proteolysis under nondenaturing conditions or are not soluble in aqueous solution. Digestion procedures performed in the presence of chaotropic agents and SDS are described, and support protocols provide instructions for preparing enzyme stocks and reducing and alkylating peptides prior to sequencing or HPLC analysis.

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