A proteome-scale study on in vivo protein Nα-acetylation using an optimized method

Protein N-terminal acetylation (Na-acetylation) is among the most common modifications in
eukaryotes. We previously described a simple method to enrich Na-modified peptides using
CNBr-activated Sepharose resin. A limitation of this method is that an optimal ratio of sample
to resin had to be determined prior to the analysis since Lys-containing Na-modified peptides
may be lost. To address this problem, we hereby present an optimized method by the
introduction of double incubation at pH 6.0. We demonstrate with the optimized method that
the Na-modified peptides can be enriched regardless of whether e-NH2 is present or not, and
the sample to resin ratio optimization is no longer necessary. Another improvement was
accomplished by the inclusion of the singly charged precursor for MS/MS fragmentation to
alleviate the shortcoming of the reduced charge state of Na-modified peptides. We employed a
duplicate experiment using 80 mg samples each and identified 922 IPI annotated and 103 IPI
unannotated acetylated N-termini from 989 proteins, so far the largest acetylated N-termini
data set acquired from a tryptic digest. Furthermore, the reproducibility of the Na-acetyl
proteome approach was evaluated and its complementarity to the regular proteome approach
was analyzed. The unexpected coupling of CNBr-activated Sepharose to His-containing
peptides via the imidazole group was discovered.