Quantitation and facilitated de novo sequencing of proteins by isotopic N-terminal labeling of peptides with a fragmentation-directing moiety

Author(s): Münchbach M, Quadroni M, Miotto G, James P

Abstract

We describe a method for comparative quantitation and de novo peptide sequencing of proteins separated either by standard chromatographic methods or by one- and two-dimensional polyacrylamide gel electrophoresis. The approach is based on the use of an isotopically labeled reagent to quantitate (by mass spectrometry) the ratio of peptides from digests of a protein being expressed under different conditions. The method allows quantitation of the changes occurring in spots or bands that contain more than one protein and has a greater dynamic range than most staining methods. Since the reagent carries a fixed positive charge under acidic conditions and labels only the N-terminal of peptides, the interpretation of tandem mass spectra to obtain sequence information is greatly simplified. The sequences can easily be extracted for homology searches instead of using indirect mass spectral-based searches and are independent of posttranslational modifications.

Similar Articles

Protein identification by mass profile fingerprinting

Author(s): James P, Quadroni M, Carafoli E, Gonnet G

Modular Peptide Synthetases Involved in Nonribosomal Peptide Synthesis

Author(s): Marahiel MA, Stachelhaus T, Mootz HD

Improving peptide fragmentation by N-terminal derivatization with high proton affinity

Author(s): Miyashita M, Hanai Y, Awane H, Yoshikawa T, Miyagawa H

Distortionless Enhancement of NMR Signalsby Polarization Transfer

Author(s): Doddrell DM, Pegg DT, Bendall MR

t-kagaku

Author(s): http://www

Curtin-Hammett and steric effects in HOBt acylation regiochemistry

Author(s): Brink BD, DeFrancisco JR, Hillner JA, Linton BR

Termination of the structural confusion between plipastatin A1 and fengycin IX

Author(s): Honma M, Tanaka K, Konno K, Tsuge K, Okuno T, et al.