Trimethylation Enhancement using Diazomethane (TrEnDi): Rapid On-Column Quaternization of Peptide Amino Groups via Reaction with Diazomethane Significantly Enhances Sensitivity in Mass Spectrometry Analyses via a Fixed, Permanent Positive Charge

Abstract

Defining cellular processes relies heavily on elucidating the temporal dynamics of proteins. To this end, mass spectrometry (MS) is an extremely valuable tool; different MS-based quantitative proteomics strategies have emerged to map protein dynamics over the course of stimuli. Herein, we disclose our novel MS-based quantitative proteomics strategy with unique analytical characteristics. By passing ethereal diazomethane over peptides on strong cation exchange resin within a microfluidic device, peptides react to contain fixed, permanent positive charges. Modified peptides display improved ionization characteristics and dissociate via tandem mass spectrometry (MS(2)) to form strong a2 fragment ion peaks. Process optimization and determination of reactive functional groups enabled a priori prediction of MS(2) fragmentation patterns for modified peptides. The strategy was tested on digested bovine serum albumin (BSA) and successfully quantified a peptide that was not observable prior to modification. Our method ionizes peptides regardless of proton affinity, thus decreasing ion suppression and permitting predictable multiple reaction monitoring (MRM)-based quantitation with improved sensitivity.

Peptides Labeled with Pyridinium Salts for Sensitive Detection and Sequencing by Electrospray Tandem Mass Spectrometry

“…Wasslen and Co 38. reported that AEFVEVTK [248–256], a tryptic fragment of BSA was not detectable in MS experiment as an unmodified compound, probably due to its low ionization efficiency or low proton affinity.…”

Click-Based Libraries of SFTI-1 Peptides: New Methods Using Reversed-Phase Silica

“…For our system, RP C18 silica was chosen to non-covalently immobilize peptide reactants, inspired by previous work using solution-solid mixed phase derivitization for proteomics. 21,22 The silica surface offers the unique conditions of site isolation of peptides, thereby disfavoring undesirable intermolecular reactions while favoring products labeled with the small molecule. In addition, the silica surface provides a unique reaction environment by co-localizing the peptide and reagents through adsorption.…”

Post‐Translational Backbone Engineering through Selenomethionine‐Mediated Incorporation of Freidinger Lactams

“…[23] Theu nique polyvalent adsorption kinetics of peptides onto reverse-phase C 18 silica allows the peptide to be adsorbed to the silica while small-molecule reagents can be added or removed through washing. [23][24][25][26] Taking advantage of the distinct differences between polyvalent versus monovalent binding kinetics, sequential reactions can be performed on an immobilized peptide while reagents can be washed away and solvents exchanged ( Figure 3). [23][24][25][26] Binding peptide substrates to reverse-phase C 18 silica allows for facile solvent transfer, introduction of small-molecule reagents,a nd removal of excess or consumed reagents,and critically allows for multiple reactions to be performed without intermediate preparative HPLC purification.…”

“…[23][24][25][26] Taking advantage of the distinct differences between polyvalent versus monovalent binding kinetics, sequential reactions can be performed on an immobilized peptide while reagents can be washed away and solvents exchanged ( Figure 3). [23][24][25][26] Binding peptide substrates to reverse-phase C 18 silica allows for facile solvent transfer, introduction of small-molecule reagents,a nd removal of excess or consumed reagents,and critically allows for multiple reactions to be performed without intermediate preparative HPLC purification. [23] Theu tilization of reverse-phase C 18 silica, which is ubiquitous for chromatographic separation in peptide laboratories,a lso allows for the determination of washing and elution conditions by RP-HPLC.…”