On-membrane tryptic digestion of proteins for mass spectrometry analysis.
Identification of proteins and characterization of posttranslational modifications are crucial steps for many biological, biochemical, and biomedical studies, and mass spectrometry has become the method of choice for these analyses. Here we describe two methods for the on-membrane digestion of proteins electroblotted onto nitrocellulose membranes prior to analysis by mass spectrometry. These on-membrane methods take approximately half the time of in-gel digestion and provide better digestion efficiency, due to the better accessibility of the protease to the proteins adsorbed onto the nitrocellulose, and better protein sequence coverage, especially for membrane proteins where large and hydrophobic peptides are commonly present.
Department of Pharmacology, Skirball Institute of Biomolecular Medicine, New York University School of Medicine, 540 First Avenue, New York, NY, 10016, USA.
Jose L
JL
Luque-Garcia
Thomas A
TA
Neubert
D000595:Amino Acid Sequence; D003101:Collodion / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis; D011506:Proteins / Q000737:chemistry / Q000378:metabolism
D010446:Peptide Fragments; D011506:Proteins; D003101:Collodion