BioProtocols--protein sequencing 1

Protein Sequencing Protocols -- (1) (2)

Protein sequencing - From Wikipedia, the free encyclopedia

Introduction: Proteins are found in every cell and are essential to every biological process, protein structure is very complex: determining a protein's structure involves first protein sequencing - determining the amino acid sequences of its constituent peptides; and also determining what conformation it adopts and whether it is complexed with any non-peptide molecules. Discovering the structures and functions of proteins in living organisms is an important tool for understanding cellular processes, and allows drugs that target specific metabolic pathways to be invented more easily.

The two major direct methods of protein sequencing are mass spectrometry and the Edman degradation reaction. It is also possible to generate an amino acid sequence from the DNA or mRNA sequence encoding the protein, if this is known. However, there are a number of other reactions which can be used to gain more limited information about protein sequences and can be used as preliminaries to the aforementioned methods of sequencing or to overcome specific inadequacies within them.

Contents

1 Determining amino acid composition

1.1 Hydrolysis

1.2 Separation

1.3 Quantitative analysis

2 N-terminal amino acid analysis

3 C-terminal amino acid analysis

4 Edman degradation

5 The Edman degradation reaction

5.1 Limitations of the Edman degradation

6 Mass spectrometry

7 Predicting protein sequence from DNA/RNA sequences

8 References

Other Protein Sequencing Protocols

SAMPLE PREPARATION TIPS FOR THE PROTEIN SEQUENCERS

(Additional Protein Sequencing Sample Preparation Tips can be found at: ABRF Home Page )

In-Gel Digest procedure for obtaining internal peptide sequences from proteins with blocked N-termini.

On-PVDF membrane digest procedure for obtaining internal peptide sequences from proteins with blocked N-termini.

N-terminal Protein/Peptide Sequencing

Carboxyl-Terminal Sequencing Methods - Shively, John

Lab-On-A-Chip.com

Matrix Sequencing: A novel method of polynucleotide analysis utilizing probes containing universal nucleotides, Saba JA, US Patent Appl 20030036073, Filed Feb 2003

Peptide Sequence Databases

nr

All non-redundant GenBank CDS translations+PDB+SwissProt+PIR+PRF

month

All new or revised GenBank CDS translation+PDB+SwissProt+PIR+PRF released in the last 30 days.

swissprot

Last major release of the SWISS-PROT protein sequence database (no updates)

Drosophila genome

Drosophila genome proteins provided by Celera and BerkeleyDrosophila Genome Project (BDGP).

yeast

Yeast (Saccharomyces cerevisiae) genomic CDS translations

ecoli

Escherichia coli genomic CDS translations

pdb

Sequences derived from the 3-dimensional structure from Brookhaven Protein Data Bank

kabat [kabatpro]

Kabat's database of sequences of immunological interest

alu

Translations of select Alu repeats from REPBASE, suitable for masking Alu repeats from query sequences. It is available by anonymous FTP from ncbi.nlm.nih.gov (under the /pub/jmc/alu directory). See "Alu alert" by Claverie and Makalowski, Nature vol. 371, page 752 (1994) .