A new mass-spectrometric C-terminal sequencing technique finds a similarity between γ-interferon and α2-interferon and identifies a proteolytically clipped γ-interferon that retains full antiviral activity

Abstract

A novel mass-spectrometric technique is described that permits the identification of the C-terminal peptide of a protein. The technique involves the incorporation of 18O into all alpha-carboxy groups liberated during enzyme-catalysed partial hydrolysis of the protein, followed by mass spectrometry to identify as the C-terminal peptide the only peptide that did not incorporate any 18O. The technique has been used to identify the true C-terminal tryptic peptide of a bacterially produced gamma-interferon and to distinguish it from a peptide produced by anomalous tryptic cleavage. It was found that a closely similar sequence segment of bacterially produced alpha 2-interferon undergoes an analogous cleavage. The technique was also used to identify the C-terminus of a clipped gamma-interferon that retains full antiviral activity.

Quantitative analysis of the low molecular weight serum proteome using 18O stable isotope labeling in a lung tumor xenograft mouse model

“…The addition of trypsin to these samples serves to mediate the exchange of two equivalents of 16 O at the carboxy-terminus of each peptide for two equivalents of 18 O in the sample reconstituted in H 2 18 O. Two complete enzyme turnovers in the presence of H 2 18 O results in a 4 Da increase in mass of each tryptic peptide [15][16][17]. The LMW proteome digestates were combined, fractionated into 96 samples using SCXLC, and each analyzed by nanoRPLC-MS/MS.…”

Quantitative mass spectrometry in proteomics: a critical review

“…An elegant and specific way to introduce an isotope label into peptides is the use of trypsin-or Glu-C-catalyzed incorporation of 18 O during protein digestion [20,21]. This has originally been employed to aid de novo sequencing of peptides by mass spectrometry [22] but has recently also been applied to quantitative proteomic applications (for a recent review see Ref. [23]).…”

Microwave‐assisted proteomics

“…Typically, large quantities of proteins are required due to the inefficiencies of the coupling, cleavage, and conversion processes. Consequently, many researchers employ alternative methods including analysis of pure sequence coverage from an LC-MS/MS experiment, the use of carboxypeptidases, anhydrotrypsin (Kumazaki, Terasawa, & Ishii, 1987), or isotopic labeling during proteolytic digestion (Rose et al, 1983). More recently, top-down proteomic techniques using either collision-activated dissociation (CAD) (Suckau & Resemann, 2003), electron capture dissociation (ECD) (Ge et al, 2002), or yielded electron-transfer dissociation (ETD) (Syka et al, 2004) have yielded predominantly N-and C-terminal fragment ions when performed on mid-size proteins.…”