3 research outputs found
High-Resolution Mapping of Carbene-Based Protein Footprints
Carbene chemistry has been used recently in structural
mass spectrometry
as a labeling method for mapping protein surfaces. The current study
presents a method for quantitating label distribution at the amino
acid level and explores the nature and basis for an earlier observation
of labeling bias. With the use of a method based on liquid chromatography–tandem
mass spectrometry (LC–MS/MS) applied to digests of holo-calmodulin,
we developed a quantitation strategy to map site-specific incorporation
of carbene, generated from photolysis of ionic label precursors 2-amino-4,4-azipentanoic
acid and 4,4-azipentanoic acid. The approach provides reliable incorporation
data for fragments generated by electron-transfer dissociation, whereas
high-energy collisional dissociation leads to energy and sequence-dependent
loss of the label as a neutral. However, both can produce data suitable
for mapping residues in the interaction of holo-calmodulin
with M13 peptide ligand. Site-specific labeling was monitored as a
function of reagent, ionic strength, and temperature, demonstrating
that electrostatic interactions at the protein surface can “steer”
the distribution of label precursors to sites of surface charge and
favor label insertion into residues in the vicinity of the surface
charge. A further preference for insertion into carboxylates was observed,
based on chemical reactivity. We suggest that decoupling surface partitioning
from the chemistry of insertion offers a flexible, tunable labeling
strategy for structural mass spectrometry that can be applied to a
broad range of protein surface compositions and promotes the design
of reagents to simplify the workflow
Carbonyl-Reactive Tandem Mass Tags for the Proteome-Wide Quantification of N-Linked Glycans
N-Linked protein glycosylation is one of the most prevalent
post-translational
modifications and is involved in essential cellular functions such
as cell–cell interactions and cellular recognition as well
as in chronic diseases. In this study, we explored stable isotope
labeled carbonyl-reactive tandem mass tags (glyco-TMTs) as a novel
approach for the quantification of N-linked glycans. Glyco-TMTs bearing
hydrazide- and aminooxy-functionalized groups were compared for glycan
reducing end derivatization efficiency and quantification merits.
Aminooxy TMTs outperform the hydrazide reagents in terms of labeling
efficiency (>95% vs 65% at 0.1 ÎĽM) and mass spectrometry
based
quantification using heavy/light-TMT labeled glycans enabled accurate
quantification in MS1 spectra (CV < 15%) over a broad dynamic range
(up to 1:40). In contrast, isobaric TMT labeling with quantification
of reporter ions in tandem mass spectra suffered from severe ratio
compression already at low sample ratios. To demonstrate the practical
utility of the developed approach, we characterized the global N-linked
glycosylation profiles of the isogenic human colon carcinoma cell
lines SW480 (primary tumor) and SW620 (metastatic tumor). The data
revealed significant down-regulation of high-mannose glycans in the
metastatic cell line
Carbonyl-Reactive Tandem Mass Tags for the Proteome-Wide Quantification of N-Linked Glycans
N-Linked protein glycosylation is one of the most prevalent
post-translational
modifications and is involved in essential cellular functions such
as cell–cell interactions and cellular recognition as well
as in chronic diseases. In this study, we explored stable isotope
labeled carbonyl-reactive tandem mass tags (glyco-TMTs) as a novel
approach for the quantification of N-linked glycans. Glyco-TMTs bearing
hydrazide- and aminooxy-functionalized groups were compared for glycan
reducing end derivatization efficiency and quantification merits.
Aminooxy TMTs outperform the hydrazide reagents in terms of labeling
efficiency (>95% vs 65% at 0.1 ÎĽM) and mass spectrometry
based
quantification using heavy/light-TMT labeled glycans enabled accurate
quantification in MS1 spectra (CV < 15%) over a broad dynamic range
(up to 1:40). In contrast, isobaric TMT labeling with quantification
of reporter ions in tandem mass spectra suffered from severe ratio
compression already at low sample ratios. To demonstrate the practical
utility of the developed approach, we characterized the global N-linked
glycosylation profiles of the isogenic human colon carcinoma cell
lines SW480 (primary tumor) and SW620 (metastatic tumor). The data
revealed significant down-regulation of high-mannose glycans in the
metastatic cell line