title: Proteomic analyses using Grifola frondosa metalloendoprotease Lys-N.
abstract: Proteomic analysis typically has been performed using proteins digested with trypsin because of the excellent fragmentation patterns they produce in collision induced dissociation (CID). For analyses in which high protein coverage is desirable, such as global monitoring of post-translational modifications, additional sequences can be seen using parallel digestion with a second enzyme. We have benchmarked a relatively obscure basidomycete-derived zinc metalloendopeptidase, Lys-N, that selectively cleaves the amide bond N-terminal of lysine residues. We have found that Lys-N digestion yields peptides with easily assigned CID spectra. Using a mixture of purified proteins as well as a complex yeast lysate, we have shown that Lys-N efficiently digests all proteins at the predicted sites of cleavage. Shotgun proteomics analyses of Lys-N digests of both the standard mixture and yeast lysate yielded peptide and protein identification numbers that were generally comparable to trypsin digestion, whereas the combination data from Lys-N and trypsin digestion substantially enhanced protein coverage. During CID fragmentation, the additional amino terminal basicity enhanced b-ion intensity which was reflected in long b-ion tags that were particularly pronounced during CID in a quadrupole. Finally, immonium ion peaks produced from Lys-N digested peptides originate from the carboxy terminus in contrast to tryptic peptides where immonium ions originate from the amino terminus.
authors.affiliation: Institute for Systems Biology, 1441 North 34th Street, Seattle, Washington 98103, University of Wisconsin, 1101 University Avenue, Madison, Wisconsin 53706, USA.
authors.forename: Laura
authors.initials: L
authors.lastname: Hohmann
authors.forename: Carly
authors.initials: C
authors.lastname: Sherwood
authors.forename: Ashley
authors.initials: A
authors.lastname: Eastham
authors.forename: Amelia
authors.initials: A
authors.lastname: Peterson
authors.forename: Jimmy K
authors.initials: JK
authors.lastname: Eng
authors.forename: James S
authors.initials: JS
authors.lastname: Eddes
authors.forename: David
authors.initials: D
authors.lastname: Shteynberg
authors.forename: Daniel B
authors.initials: DB
authors.lastname: Martin
mesh_terms: D005656:Fungal Proteins / Q000378:metabolism; D048691:Grifola / Q000201:enzymology*; D008239:Lysine / Q000378:metabolism*; D045727:Metalloexopeptidases / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism*; D020543:Proteome / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D010455:Peptides; D020543:Proteome; D045727:Metalloexopeptidases; D014357:Trypsin; D008239:Lysine