Selective enrichment of azide-containing peptides from complex mixtures

Abstract

A general method is described to sequester peptides containing azides from complex peptide mixtures, aimed at facilitating mass spectrometric analysis to study different aspects of proteome dynamics. The enrichment method is based on covalent capture of azide-containing peptides by the azide-reactive cyclooctyne (ARCO) resin and is demonstrated for two different applications. Enrichment of peptides derived from cytochrome c treated with the azide-containing cross-linker bis(succinimidyl)-3-azidomethyl glutarate (BAMG) shows several cross-link containing peptides. Sequestration of peptides derived from an Escherichia coli proteome, pulse labeled with the bio-orthogonal amino acid azidohomoalanine as substitute for methionine, allows identification of numerous newly synthesized proteins. Furthermore, the method is found to be very specific, as after enrichment over 87% of all peptides contain (modified) azidohomoalanine.

Figure 1. Overview of protocol and reagents for enrichment of azide-containing peptides

To label newly synthesized proteins, methionine auxotrophic E. coli K12 cells are grown on a medium where methionine has been replaced by azidohomoalanine, incorporating this non-canonical amino acid into proteins. To obtain spatial distance information, proteins are cross-linked with bis(succinimidyl)-3-azidomethyl glutarate (BAMG). After isolation or cross-linking, proteins are digested and the obtained peptide mixture is incubated with the ARCO-resin. After cleavage from the resin, enriched peptides are analysed by LC-MS/MS. a, Workflow of the enrichment method. b, Azidohomoalanine (azhal) is a methionine analogue, containing an azide group instead of a thiomethoxy group. c, Structure of the azide-containing cross-linker BAMG. d, ARCO-resin, consists of a poly-dimethylacrylamide solid support, a disulfide as cleavable linker and a cyclooctyne as reactive group towards azides. Via the strain-promoted (3+2) cycloaddition azide-containing peptides are captured on the resin. e, Product after enrichment, peptides are modified with the cyclooctyne and linker, adding 358.2 Da in mass.

Figure 2. Enrichment of a single model peptide shows the selective reaction and release of the peptide with the ARCO-resin

A model peptide containing a single azhal residue was incubated for 24 hours at 40 °C with the ARCO-resin. a, Peptide before addition to the ARCO-resin. b, Flow through (ft) after 24h incubation at 40 °C c, Enriched peptide (ep). d, Sequence of model peptide used to test and optimize the enrichment method. X: azhal. N-terminus is acetylated. ◆: loss of N₂ and uptake of 2 H. *: salt adducts.

Figure 3. Quantification of enrichment of a model peptide at room temperature and 40 °C for different incubation times

MALDI-TOF spectra of quantification experiments. a, Enrichment experiment at room temperature. b, Enrichment experiment at 40 °C. c, Control. H₃: Ac(H₃) Pan016, flow through/enriched peptide. D₃: Ac(D₃)-Pan016, reference peptide.

Figure 4. Sequence coverage of labeled Photoactive Yellow Protein (AzPYP) before and after enrichment

In this figure the peptides identified by both MALDI-TOF and LC-Q-TOF-MS/MS are depicted in the sequence of PYP, a protein consisting of 142 amino acids. It contains six methionine residues, which have been replaced by azhal (X, marked in figure by ▼). The cysteine residue has been marked by ■.

Figure 5. Enrichment of cross-linked peptides from cytochrome c cross-linked with BAMG

MALDI-TOF spectra of the tryptic digest of cytochrome c cross-linked with BAMG a, before and b, after enrichment. ◇: Unmodified peptide. ◀: Cross-link containing peptide. ■: Cross-link containing peptide observed before and after enrichment.

Figure 6. Fragmentation spectrum shows the fragmentation behavior of modified peptide after sequestration

a, Fragmentation spectrum of a peptide belonging to 5, 10-methylenentetrahydrofolate reductase enriched from a labeled E.coli proteome is shown. FADX(mod)TNVR at m/z 1306.44. b, Overview of observed and assigned fragments in peptide sequence. y*: loss of the cyclooctyne with the linker. ◀: cyclooctyne with linker with m/z 402.2. ▼: further fragmentation of the cyclooctyne gives rise to a benzoylium ion with 268.1 Da.