A review of COFRADIC techniques targeting protein N-terminal acetylation

Abstract

Acetylation of nascent protein Nalpha-termini is a common modification among archae and eukaryotes and can influence the structure and function of target proteins. This modification has been studied on an individual protein or (synthetic) peptide level or on a proteome scale using two-dimensional polyacrylamide gel electrophoresis. We recently developed mass spectrometry driven proteome analytical approaches specifically targeting the amino (N) terminus of proteins based on the concept of diagonal reverse-phase chromatography. We here review how this so-called combined fractional diagonal chromatography (COFRADIC) technique can be used in combination with differential mass-tagging strategies as to both qualitatively and quantitatively assess protein Nalpha-acetylation in whole proteomes.

Figure 1

General work-flow of the N-terminal COFRADIC procedure.

Figure 2

A, B and C: Representative MS spectra of N-terminal peptides from partially alpha-N-acetylated proteins. The MS-spectrum from the N-terminal peptide (doubly charged precursor) of the DnaJ homolog subfamily C member 2 (¹MLLLPSAADGR¹¹, 44% acetylated) reveals two distinguishable isotopic envelopes of the two isotopomers (i.e. the acetylated (Ac) and trideutero-acetylated forms (AcD₃)) (A). Panels B & C show MS-spectra of the N-terminal peptide (triply charged precursor) of the signal recognition particle 68 kDa protein (²AAEKQVPGGGGGGGSGGGGGSGGGGSGGGR³¹) when trideutero-acetylated (B) or ¹³C₂D₃-acetylated (C) (53% acetylated). Panel B illustrates the need for increased mass spacing by heavier isotopomers. D: Comparison of protein N-trideuteroacetylation and N-propionylation. A protein that is partially in vivo α-N-acetylated can be modified in vitro by N-trideutero-acetylation or N-propionylation. When trideutero-acetylation is used, the RP-HPLC elution profiles of the α-N-acetylated and α-N-trideutero-acetylated variants are indistinguishable and the peptide variants only segregate upon MS analysis by their 3 Da mass difference. In contrast, when N-propionylation is used the α-N-acetylated and α-N-propionylated variants segregate upon RP-HPLC, with the propionylated variant eluting at a later time because of increased hydrophobicity. E.: The use of methionine oxidation to segregate methionine-containing from non-methionine-containing N-terminal peptides. Methionine oxidation, when applied in between the primary and secondary RP-HPLC separations and after TNBS modification, causes methionine-sulfoxide containing N-terminal peptides to shift to earlier elution times on RP-HPLC. As a result the N-terminal peptides of NatB (Met-Asn, Met-Asp and Met-Glu) and NatC (Met-Ile, Met-Leu and Met-Phe) substrates, as well as of substrates of yet unidentified NATs (e.g. Met-Lys) migrate out of the primary collection intervals and are in this way enriched from the non-methionine-containing NatA substrates.