Detectability of Biotin Tags by LC–MS/MS

Abstract

The high affinity of biotin to streptavidin has made it one of the most widely used affinity tags in proteomics. Early methods used biotin for enrichment alone and mostly ignored the biotin labeled peptide. Recent advances in labeling led to an increase in biotinylation efficiency and shifted the interest to detection of the site of biotinylation. This increased confidence in identification and provides additional structural information yet it requires efficient release of the biotinylated protein/peptide and sensitive separation and detection of biotinylated peptides by LC-MS/MS. Despite its long use in affinity proteomics the effect of biotinylation on the chromatographic, ionization, and fragmentation behaviour and ultimate detection of peptides is not well understood. To address this we compare two commercially-available biotin labels EZ-Link Sulfo-NHS-Biotin and Sulfo-NHS-SS-Biotin, the latter one containing a labile linker to efficiently release biotin to determine the effects of peptide modification on peptide detection. We describe an increase of hydrophobicity and charge reduction with increasing number of biotin labels attached. Based on our data we recommend gradient optimization to account for more hydrophobic biotinylated peptides and include singly charged precursors to account for charge reduction by biotin.

Benchmarking Cleavable Biotin Tags for Peptide-Centric Chemoproteomics

“…Although antibody-based approaches have been recently introduced to enrich biotin-modified peptides, 5,6 MS analysis of these peptides may be compromised by their poor efficiency in ionization and fragmentation. 7 To address these limitations, diverse chemical moieties have been implemented for developing azide biotin reagents bearing cleavable tags, which allow release of labeled proteins/peptides under selective and relatively mild conditions, and yield modified peptides without the biotin moiety. 8,9 In general, three typical chemoproteomic workflows have been developed by incorporating with these reagents (Figure 1A and Figure S1).…”

“…The direct identification of probe/tag-modified peptides (herein termed peptide-centric) not only provides definitive confirmation but also generates invaluable information for prioritizing follow-up functional analysis. Although antibody-based approaches have been recently introduced to enrich biotin-modified peptides, , MS analysis of these peptides may be compromised by their poor efficiency in ionization and fragmentation …”

Ycs4 Subunit of Saccharomyces cerevisiae Condensin Binds DNA and Modulates the Enzyme Turnover

“…The lysines that were labeled in the Ycs4-BY complex but remained protected in the presence of DNA were marked with ruby red and salmon red in the figure. The ruby red-colored lysines appeared unlabeled in the mass spectra of the Ycs4-BY-DNA complex, whereas the salmon red-colored lysines did not appear in the mass spectrometric measurement of the Ycs4-BY-DNA, presumably due to the poor ionization of the unbiotinylated form of respective peptides . In principle, the differentially detectable peptides are not necessarily differentially labeled but are only likely to be.…”

“…The ruby red-colored lysines appeared unlabeled in the mass spectra of the Ycs4-BY-DNA complex, whereas the salmon red-colored lysines did not appear in the mass spectrometric measurement of the Ycs4-BY-DNA, presumably due to the poor ionization of the unbiotinylated form of respective peptides. 40 In principle, the differentially detectable peptides are not necessarily differentially labeled but are only likely to be. Therefore, the two types of lysines were marked in different colors in Figure 2 and thereafter.…”

Comprehensive Discovery of the Accessible Primary Amino Group-Containing Segments from Cell Surface Proteins by Fine-Tuning a High-Throughput Biotinylation Method

“…Additionally, the labeling may decrease the charge state of the peptide precursors because it modifies the amino group side chain of lysine residues. It has been observed that the detectability of similar biotin tags containing peptides can be promoted by chromatography gradient optimization and/or by taking into account singly-charged precursors in the tandem mass spectrometry analysis [52]. We hypothesize that these factors may affect the yields of the labeled peptides/proteins from our approach.…”