Combined in-gel tryptic digestion and CNBr cleavage for the generation of peptide maps of an integral membrane protein with MALDI-TOF mass spectrometry

Bart A. van Montfort, Mark K. Doeven, Benito Canas, Liesbeth M. Veenhoff, Bert Poolman, George T. Robillard

OnderzoeksoutputAcademic

58 Citaten (Scopus)
225 Downloads (Pure)

Samenvatting

A limitation of the in-gel approaches for the generation of peptides of membrane proteins is the size and hydrophobicity of the fragments generated. For membrane proteins like the lactose transporter (LacS) of Streptococcus thermophilus, tryptic digestion or CNBr cleavage yields several hydrophobic fragments larger than 3.5 kDa. As a result, the sequence coverage of the membrane domain is low when the in-gel tryptic-digested or CNBr-cleaved fragments are analyzed by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry (MS). The combination of tryptic digestion and subsequent CNBr cleavage on the same gel pieces containing LacS approximately doubled the coverage of the hydrophobic membrane domain compared to the individual cleavage methods, while the coverage of the soluble domain remained complete. The fragments formed are predominantly below m/z 2500, which allows accurate mass measurement.
Originele taal-2English
Pagina's (van-tot)111 - 115
Aantal pagina's5
TijdschriftBiochimica et Biophysica Acta-Bioenergetics
Volume1555
Nummer van het tijdschrift1-3
DOI's
StatusPublished - 2002

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