To study the structures of proteins and protein complexes by mass spectrometry we utilize small and agile reagents capable of forming covalent bonds. As we are not restricted in abundance, enrichment strategies are employed to pull sub-stoichiometric protein (-events) into view.
The most basic, versatile, and widely used approaches for labeling peptides and proteins typically involve chemical groups capable of reacting with primary amines (-NH2). These amines are found at the N-terminus of each polypeptide chain and in the side-chain of lysine (Lys, K) amino acid residues. Primary amines are positively charged at physiological pH and thus tend to occur predominantly on the surface of native protein tertiary structures, where they are easily accessible to conjugation reagents.
We utilize the most reactive group – NHS-ester – to perform conjugation.
The crosslinking reagents we utilize carry two NHS-esters separated by a spacer arm. When these reagents react within a protein (-complex) structure, they are capable of forming a covalent bond between two residues in close proximity.
After fairly simple sample preparation steps (reduction, alkylation, and digestion) three products are obtained. (1) Normal peptides – not conjugated by the reagent and structurally not informative. (2) Mono-links – conjugated by the reagent that quenched on the other end with water or Tris and structurally of limited value. (3) Crosslinked peptide pairs – two peptides covalently bound by the reagent and structurally informative.
The crosslinked peptide pairs can be identified through shotgun proteomics, for which we developed the data analysis software pipeline XlinkX/PD.
Crosslinking mass spectrometry is however a challenging technique. Upon investigation of the abundance of the 3 products, we found that the informative crosslinked peptide pairs make up less than 0.001% of what is injected on the mass spectrometer. A tall order for any analytical technique.
Our reaction to this was to develop the reagent PhoX. At its core this is a classic reagent with two NHS-esters connected with a spacer arm. At the middle we however extended the molecule with an enrichable handle (middle-bottom) – a phosphonic acid.
The main advantage of using a phosphonic acid is that it is rock stable, yielding excellent fragmentation spectra, and is unaffected by phosphatase treatment. This allows for the selective removal of phosphorylation on peptides during the sample preparation, while leaving the reagent unaffected.
With the phosphonic acid we can use standardized IMAC (Immobilized Metal Affinity Chromatography) to enrich the sub-stoichiometric crosslinked peptide pairs from the high background of non-informative peptides. As we use the well developed IMAC enrichment technique, the enrichment step can be fully automated on sample preparation robots.
This brings them within easy reach of our mass spectrometry platforms.
The study of post-translational modifications likewise suffers from sub-stoichiometry problems. A classic example is phosphorylation, for which IMAC enrichment was developed. Based on the success of our IMAC enrichable reagent PhoX we are tackling new PTMs.