Complete Chemical Modification of Amine and Acid Functional Groups of Peptides and Small Proteins

Abstract

Summary The chemical modification of protein thiols by reduction and alkylation is common in the preparation of proteomic samples for analysis by mass spectrometry (MS). Modification at other functional groups has received less attention in MS-based proteomics. Amine modification (Lys, N-termini) by reductive dimethylation or by acylation (e.g. iTRAQ labeling) has recently gained some popularity in peptide-based approaches (bottom-up MS). Modification at acidic groups (Asp, Glu, C-termini) has been explored very minimally. Here, we describe a sequential labeling strategy that enables complete modification of thiols, amines, and acids on peptides or small intact proteins. This method includes (1) the reduction and alkylation of thiols, (2) the reductive dimethylation of amines, and (3) the amidation of acids with any of several amines. This chemical modification scheme offers several options both for the incorporation of stable isotopes for relative quantification and for improving peptides or proteins as MS analytes.

In Vitro Biosynthesis and Substrate Tolerance of the Plantazolicin Family of Natural Products

“…Though the methyltransferase has significant substrate selectivity owing to its narrow substrate-binding channel (17), all of the PZN variants used in these reactions contained both the N -terminal Arg and at least one azole, which is minimally necessary for robust methyltransferase activity. Although enzymatic dimethylation was successful, more robust modification was achieved by reductive alkylation with formaldehyde and borane-pyridine (Supplementary Figure 44) (58). Regardless of the method of dimethylation, this sequence of reactions enabled the production of a new collection of PZN variants beyond what was previously achievable in vivo (41).…”

“…Chemical dimethylation was performed using <1 mM peptide (exact concentration not known), 200 mM formaldehyde, and 300 mM borane-pyridine in 10 mM NH 4 CO 3 with 50% MeOH, for 18 h at 22 °C (58). …”

Chemical Derivatization of Peptide Carboxyl Groups for Highly Efficient Electron Transfer Dissociation

“…The latter approach addresses the issue that in many cases, peptides remain unobserved due to their chemical composition (sequence), which can cause poor chromatographic elution, poor ionization, and/or poor fragmentation. We have previously reported a robust and efficient derivitization chemistry for the modification of protein or peptide carboxyl groups with tertiary or quaternary amines [2]. These added amines impart increased charge during positive mode electrospray ionization mass spectrometry.…”

Solution-phase and solid-phase sequential, selective modification of side chains in KDYWEC and KDYWE as models for usage in single-molecule protein sequencing

“…Highly efficient, global modification using (7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate ( PyAOP ) and N-methylmorpholine ( NMM ) has been reported. 6 Peptide 5, amidated with benzylamine dissolved in MeCN/H 2 O mixtures, and purification of the resulting modified peptides was possible. Yield for peptide 5 was 29%.…”

“…5 Cross-modification of threonine, serine, and tyrosine can occur with acylation and alkylating conditions. 6 Recently, peptide amines have been modified via reductive methylation preventing cross-reactivity with alcohol and phenol residues. Once these amines were modified, the Smith group achieved global modification of aspartate and glutamate via amidation with amine-containing compounds.…”