MdCDPM: A Mass Defect-Based Chemical-Directed Proteomics Method for Targeted Analysis of Intact Sialylglycopeptides

Protein sialylation
is ubiquitous and essential in a wide range
of biological processes. Herein, a mass defect-based chemical-directed
proteomics method (MdCDPM) was presented for targeted analysis of
intact sialylglycopeptides (SGPs). The process starts by specific
oxidation of dihydroxy in sialic acid to aldehyde, which was then
chemically labeled by two arginine isotopologues (Arg-15N4 and Arg-D4, differs by 36 mDa). The equally
mixed precursor partners, spacing tens of mDa apart, enable the direct
recognition of SGPs in MS1 level and benefit the subsequent targeted
MS2 characterization. The mass envelope of two labeled forms falling
into a narrow m/z window strengthens
recognition uniqueness greatly, and the proposed 1:1 intensity ratio
of doublets will not be readily distorted. More important, such subtle
mass differences permit multiple sialic acids labeling without additional
complexity of precursor patterns. Also, the partner m/z shifts detail the number of sialic acids contained
in the precursor species. By applying MdCDPM, femtomole quantities
of SGPs could be detected from total cell lysates, even at a signal-to-noise
ratio of as low as 3:1. In addition, assays were performed to estimate
the false positive rate and demonstrated high confidence of MdCDPM.
Furthermore, it was designed and successfully exploited to analyze
SGPs in human serum, which highlighted the feasibility of this strategy
for biological applications.