Chemical cross‐linking and mass spectrometry to map three‐dimensional protein structures and protein–protein interactions

Abstract

Closely related to studying the function of a protein is the analysis of its three-dimensional structure and the identification of interaction sites with its binding partners. An alternative approach to the high-resolution methods for three-dimensional protein structure analysis, such as X-ray crystallography and NMR spectroscopy, consists of covalently connecting two functional groups of the protein(s) under investigation. The location of the created cross-links imposes a distance constraint on the location of the respective side chains and allows one to draw conclusions on the three-dimensional structure of the protein or a protein complex. Recently, chemical cross-linking of proteins has been combined with a mass spectrometric analysis of the created cross-linked products. This review article describes the most popular cross-linking reagents for protein structure analysis and gives an overview of the different available strategies that employ chemical cross-linking and different mass spectrometric techniques. The challenges for mass spectrometry caused by the enormous complexity of the cross-linking reaction mixtures are emphasized. The various approaches described in the literature to facilitate the mass spectrometric detection of cross-linked products as well as computer software for data analyses are reviewed.

Protein Surface Mapping Using Diethylpyrocarbonate with Mass Spectrometric Detection

“…12 Figure 1 shows two example MS/MS spectra indicating that modifications can be readily determined with single amino acid resolution. For example, unmodified y 3 , y 4 , y 5 , and y 6 product ions along with modified y 7 and y 8 ions indicate that His13 is the modified residue in the Ser11-Lys19 fragment of β2m ( Figure 1a). Similarly, unmodified b ions from b 8 through b 14 , a modified b 15 ion, and a complete series of modified y ions from y 2 to y 13 indicate that Ser117 is the modified residue in the Tyr103-Lys119 fragment of myoglobin ( Figure 1b).…”

Probing the transient interaction between the small heat-shock protein Hsp21 and a model substrate protein using crosslinking mass spectrometry

“…We have used crosslinking mass spectrometry, i.e., the mass spectrometric detection of crosslinked peptides, to investigate crosslinks between proteins. This approach (also called CXMS) is currently maturing into a useful approach to characterize proteinprotein interactions, even though some technical obstacles still prevent its exploitation to its full potential (Leitner et al 2010;Singh et al 2010;Sinz 2006). Our previously developed workflow, using lysine-specific crosslinking, offline nanoliquid chromatography matrix-assisted laser desorption/ionization tandem time-of-flight (LC MALDI-TOF/TOF) mass spectrometry and an in-house developed program FINDX, was shown to allow the identification of true crosslinks that are all within crosslinking distance (<20 Å), mapping well into the structure of a protein oligomer (Lambert et al 2011b;Söderberg et al 2012).…”

‘Fixed charge’ chemical derivatization and data dependant multistage tandem mass spectrometry for mapping protein surface residue accessibility

“…However, limitations of HDX that can hamper determination of the precise location of exchange sites and therefore limit the spatial resolution that can be obtained, are the back-exchange of deuterium to hydrogen during proteolysis and sample handling before MS analysis [7,8], and hydrogen/deuterium 'scrambling' during CID-MS/MS analysis [9]. Recently, the latter problem has been largely overcome by the use of alternate dissociation techniques such as electron capture dissociation (ECD) [10] and electron-transfer dissociation (ETD) during MS/MS [11].Stable chemical labeling strategies have included (1) nonspecific oxidative modification of protein side-chain functional groups induced by hydroxyl radicals formed via the radiolysis of water by synchrotron X-ray pulses [4], or by photolysis of H 2 O 2 using pulsed UV lasers [5], and (2) specific labeling [6] or cross-linking [12] of Address reprint requests to Dr. …”