N-terminal enrichment: developing a protocol to detect specific proteolytic fragments.
Proteolytic processing events are essential to physiological processes such as reproduction, development, and host responses, as well as regulating proteins in cancer; therefore, there is a significant need to develop robust approaches for characterizing such events. The current mass spectrometry (MS)-based proteomics techniques employs a "bottom-up" strategy, which does not allow for identification of different proteolytic proteins since the strategy measures all the small peptides from any given protein. The aim of this development is to enable the effective identification of specific proteolytic fragments. The protocol utilizes an acetylation reaction to block the N-termini of a protein, as well as any lysine residues. Following digestion, N-terminal peptides are enriched by removing peptides that contain free amines, using amine-reactive silica-bond succinic anhydride beads. The resulting enriched sample has one N-terminal peptide per protein, which reduces sample complexity and allows for increased analytical sensitivity compared to global proteomics.(1) We initially compared the peptide identification and efficiency of blocking lysine using acetic anhydride (a 42 Da modification) or propionic anhydride (a 56 Da modification) in our protocol. Both chemical reactions resulted in comparable peptide identifications and approximately 95 percent efficiency for blocking lysine residues. However, the use of propionic anhydride allowed us to distinguish in vivo acetylated peptides from chemically-tagged peptides.(2) In an initial experiment using mouse plasma, we were able to identify >300 unique N-termini peptides, as well as many known cleavage sites. This protocol holds potential for uncovering new information related to proteolytic pathways, which will assist our understanding about cancer biology and efforts to identify potential biomarkers for various diseases.
Schepmoes
Athena A
AA
Pacific Northwest National Laboratory, Richland, Washington 99352, USA. athena.schepmoes@pnl.gov
Zhang
Qibin
Q
Petritis
Brianne O
BO
Qian
Wei-Jun
WJ
Smith
Richard D
RD
D000107:Acetylation; D000812:Anhydrides / Q000737:chemistry; D000818:Animals; D001709:Biotechnology / Q000379:methods; D002853:Chromatography, Liquid / Q000379:methods; D066246:ErbB Receptors / Q000737:chemistry; D006801:Humans; D008239:Lysine / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods; D010455:Peptides / Q000737:chemistry; D011422:Propionates / Q000737:chemistry; D017434:Protein Structure, Tertiary; D040901:Proteomics / Q000379:methods; D020539:Sequence Analysis, Protein / Q000379:methods*; D013387:Succinic Anhydrides / Q000737:chemistry
D000812:Anhydrides; D010455:Peptides; D011422:Propionates; D013387:Succinic Anhydrides; C031801:succinic anhydride; C096126:propionic anhydride; D066246:ErbB Receptors; D008239:Lysine