Quantitative Photo-crosslinking Mass Spectrometry Revealing Protein Structure Response to Environmental Changes

Abstract

Protein structures respond to changes in their chemical and physical environment. However, studying such conformational changes is notoriously difficult, as many structural biology techniques are also affected by these parameters. Here, the use of photo-crosslinking, coupled with quantitative crosslinking mass spectrometry (QCLMS), offers an opportunity, since the reactivity of photo-crosslinkers is unaffected by changes in environmental parameters. In this study, we introduce a workflow combining photo-crosslinking using sulfosuccinimidyl 4,4′-azipentanoate (sulfo-SDA) with our recently developed data-independent acquisition (DIA)-QCLMS. This novel photo-DIA-QCLMS approach is then used to quantify pH-dependent conformational changes in human serum albumin (HSA) and cytochrome C by monitoring crosslink abundances as a function of pH. Both proteins show pH-dependent conformational changes resulting in acidic and alkaline transitions. 93% and 95% of unique residue pairs (URP) were quantifiable across triplicates for HSA and cytochrome C, respectively. Abundance changes of URPs and hence conformational changes of both proteins were visualized using hierarchical clustering. For HSA we distinguished the N–F and the N–B form from the native conformation. In addition, we observed for cytochrome C acidic and basic conformations. In conclusion, our photo-DIA-QCLMS approach distinguished pH-dependent conformers of both proteins.

A protocol for studying structural dynamics of proteins by quantitative crosslinking mass spectrometry and data-independent acquisition

“…DIA-QCLMS improves on DDA based quantitation data [18] and provides higher coverage and fewer missing values [17]. Currently, just a few studies have been published using DIA in conjunction with crosslinking [18,53,54]. DIA-QCLMS is capable of detecting changing abundances of crosslinked peptides, even with the ratio compression encountered with increased sample complexity [18].…”

“…DIA-QCLMS is capable of detecting changing abundances of crosslinked peptides, even with the ratio compression encountered with increased sample complexity [18]. In combination with photoactivatable crosslinkers, DIA-QCLMS (photo-DIA-QCLMS) was able to distinguish pH-dependent conformers of Human serum albumin and Cytochrome C [54]. Although DIA-QCLMS has widened the scope of quantitative crosslinking in structural biology, it has been restricted by a lack of software tools supporting DIA crosslink data analysis.…”

“…c: Volcano plot after performing a two sided t-test of triplicate datasets of pH 7 and pH 10 using p-value cutoff of 0.05 and fold change cutoff of 1 (blue: significantly changing unique residue pairs for pH 10, green: significantly changing residue pairs for pH 7, grey: residue pairs that have no significant change). d: cluster plot adapted and modified from [54] showing median abundances of URPs and statistically significant shifts as a function of pH (p<0.05)(red: pH 7, green: pH 10).…”

Interfaces with Structure Dynamics of the Workhorses from Cells Revealed through Cross-Linking Mass Spectrometry (CLMS)

“…Quantitative cross-linking mass spectrometry (QCLMS) approaches investigate protein structures as well as the dynamics of their interactions [ 46 , 47 , 48 , 49 , 50 , 51 ]. QCLMS is often performed using a unique cross-linker that introduces a corresponding mass shift after isotope labeling specific only to the cross-linked peptides [ 46 , 47 , 50 , 52 , 53 ], followed by quantitation of cross-links in MS1.…”

“…The extraction of cross-linked peptide quantities from DIA data is usually performed utilizing a spectral library prepared particularly from investigated samples. Muller et al developed a novel DIA-QCLMS approach utilizing photo activatable cross-linkers ensuring reliable quantitation of cross-linked proteins across a wide range of environmental changes, such as pH, temperature pressure, or concentration [ 51 , 52 , 57 ]. DIA approaches promise to be useful in future applications of quantitative cross-linking proteomics, due to their precision, reproducibility, and label-free manner.…”