Abstract
Mass spectrometric analysis of trace amounts of peptides may be problematic due to the insufficient ionization efficiency resulting in limited sensitivity. One of the possible ways to overcome this problem is the application of ionization enhancers. Herein we developed new ionization markers based on 2,4,6-triphenylpyridinium and 2,4,6-trimethylpyridinium salts. Using of inexpensive and commercially available pyrylium salt allows selective derivatization of primary amino groups, especially those sterically unhindered, such as ε-amino group of lysine. The 2,4,6-triphenylpyridinium modified peptides generate in MS/MS experiments an abundant protonated 2,4,6-triphenylpyridinium ion. This fragment is a promising reporter ion for the multiple reactions monitoring (MRM) analysis. In addition, the fixed positive charge of the pyridinium group enhances the ionization efficiency. Other advantages of the proposed ionization enhancers are the simplicity of derivatization of peptides and the possibility of convenient incorporation of isotopic labels into derivatized peptides.
Highlights
Elucidation of processes taking place in living cells, especially interactions of diverse range of biomolecules, is an object of widespread interest[1,2]
Mass spectrometry analysis of trace amounts of peptides is problematic due to the insufficient ionization efficiency, which results in limited sensitivity
We describe an application of 2,4,6-trisubstituted pyrylium salts as reagents allowing incorporation of pyridinium moiety to amino group of a peptide and studies of the effect of this modification on the ionization efficiency
Summary
The proton affinity is an important parameter influencing the intensity, which in turn depends not necessarily on the concentration, but on the specific characteristics of a peptide sequence, abundant signals of Arg-containing peptides in ESI spectra[10] Another way to enhance ionization efficiency of peptides is their derivatization which results in a formation of fixed charged peptide derivative and their increased intensity in ESI11,12. We present a new approach of peptide derivatization based on conversion of ε-amino groups of lysine side chain to trisubstituted pyridinium derivative This derivatization results in a highly improved sensitivity of peptide detection. We presented the straightforward and cost-effective synthesis of deuterated isotopologues This fact, combined with the elimination of the pyridinium reporter ion, enables highly sensitive MRM-based analysis, which is potentially useful in quantitative proteomics
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