Utility of formaldehyde cross‐linking and mass spectrometry in the study of protein–protein interactions

Abstract

For decades, formaldehyde has been routinely used to cross-link proteins in cells, tissue, and in some instances, even entire organisms. Due to its small size, formaldehyde can readily permeate cell walls and membranes, resulting in efficient cross-linking, i.e. the formation of covalent bonds between proteins, DNA, and other reactive molecules. Indeed, formaldehyde cross-linking is an instrumental component of many mainstream analytical/cell biology techniques including chromatin immunoprecipitation (ChIP) of protein-DNA complexes found in nuclei; immunohistological analysis of protein expression and localization within cells, tissues, and organs; and mass spectrometry (MS)-compatible silver-staining methodologies used to visualize low abundance proteins in polyacrylamide gels. However, despite its exquisite suitability for use in the analysis of protein environments within cells, formaldehyde has yet to be commonly employed in the directed analysis of protein-protein interactions and cellular networks. The general purpose of this article is to discuss recent advancements in the use of formaldehyde cross-linking in combination with MS-based methodologies. Key advantages and limitations to the use of formaldehyde over other cross-linkers and technologies currently used to study protein-protein interactions are highlighted, and formaldehyde-based experimental approaches that are proving very promising in their ability to accurately and efficiently identify novel protein-protein and multiprotein interaction complexes are presented.

Extraction of extracellular polymeric substances (EPS) from anaerobic granular sludges: comparison of chemical and physical extraction protocols

“…The association of formaldehyde with NaOH, which leads to the highest extraction yields, seems to be the most efficient extraction procedure. Formaldehyde itself binds to the cell by reacting with functional groups like carbonyl, amino, or hydroxyl groups (Sutherland et al 2008). Once fixed to the membrane, it acts as a buffer that prevents cell lysis.…”

“…For formaldehyde combined with a heating step, extraction yields are higher than the heating extraction alone. Addition of formaldehyde, which is considered to protect the cell membrane during extraction (Sutherland et al 2008), seems also to increase the EPS extraction yield. Formaldehyde can react with amino groups of proteins or aminopolysaccharides (Comte et al 2006a) to alkylate cell wall molecules or EPS and thus destabilizes the structure of granules and increases the release of EPS.…”

Avirulence Protein 3a (AVR3a) from the Potato Pathogen Phytophthora infestans Forms Homodimers through Its Predicted Translocation Region and Does Not Specifically Bind Phospholipids

“…At ambient temperature, formaldehyde forms predominantly immonium cations that are reactive toward nucleophiles, such as the N terminus of a protein and the lateral chains of cysteine, lysine, histidine, and tyrosine residues (14). Formaldehyde is ideal to detect specific protein-protein interactions due to (i) a short reaction time that minimizes nonspecific cross-linking, (ii) the formation of highly reactive intermediates that allow fixation of transient interactions, and (iii) a short cross-linking distance (ϳ0.2-0.3 nm) (15). Cross-linking conditions were optimized for AVR3a(22-147)-His 6 at room temperature.…”

Identification and characterization of posttranslational modification-specific binding proteins in vivo by mammalian tethered catalysis

“…Also, stable cell lines containing the experimental and control MTeC bait plasmids could be created; thereby, providing a virtually unlimited pool of input material to identify PTMBPs that are in low abundance. Also, the use of cross-linking reagents could also be used before the purification process to capture PTMBPs that transiently bind the modified MTeC bait fusion protein (24). A more modern and sophisticated way to avoid both of these potential problems would be to use the stable isotope labeling with amino acids method (SILAC) followed by MS to identify proteins specifically associated with the experimental MTeC sample, but absent in control samples (25).…”