title: Large-scale quantitative assessment of different in-solution protein digestion protocols reveals superior cleavage efficiency of tandem Lys-C/trypsin proteolysis over trypsin digestion.
abstract: The complete and specific proteolytic cleavage of protein samples into peptides is crucial for the success of every shotgun LC-MS/MS experiment. In particular, popular peptide-based label-free and targeted mass spectrometry approaches rely on efficient generation of fully cleaved peptides to ensure accurate and sensitive protein quantification. In contrast to previous studies, we globally and quantitatively assessed the efficiency of different digestion strategies using a yeast cell lysate, label-free quantification, and statistical analysis. Digestion conditions include double tryptic, surfactant-assisted, and tandem-combinatorial Lys-C/trypsin digestion. In comparison to tryptic digests, Lys-C/trypsin digests were found most efficient to yield fully cleaved peptides while reducing the abundance of miscleaved peptides. Subsequent sequence context analysis revealed improved digestion performances of Lys-C/trypsin for miscleaved sequence stretches flanked by charged basic and particulary acidic residues. Furthermore, targeted MS analysis demonstrated a more comprehensive protein cleavage only after Lys-C/trypsin digestion, resulting in a more accurrate absolute protein quantification and extending the number of peptides suitable for SRM assay development. Therefore, we conclude that a serial Lys-C/trypsin digestion is highly attractive for most applications in quantitative MS-based proteomics building on in-solution digestion schemes.
authors.affiliation: Proteomics Core Facility, Biozentrum, Basel University, Basel, Switzerland.
authors.forename: Timo
authors.initials: T
authors.lastname: Glatter
authors.forename: Christina
authors.initials: C
authors.lastname: Ludwig
authors.forename: Erik
authors.initials: E
authors.lastname: Ahrné
authors.forename: Ruedi
authors.initials: R
authors.lastname: Aebersold
authors.forename: Albert J R
authors.initials: AJ
authors.lastname: Heck
authors.forename: Alexander
authors.initials: A
authors.lastname: Schmidt
mesh_terms: D057230:Limit of Detection; D008239:Lysine / Q000378:metabolism*; D059748:Proteolysis; D015203:Reproducibility of Results; D012996:Solutions; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000378:metabolism*
chemical_list: D012996:Solutions; D014357:Trypsin; D008239:Lysine