Combining a NHS ester and glutaraldehyde improves crosslinking prior to MALDI MS analysis of intact protein complexes

Abstract

Protein complexes play pivotal roles in cellular life. Nevertheless, their characterization remains a substantial challenge. Mass spectrometry (MS) is an emerging tool to study protein assemblies, and electrospray ionization (ESI) is often used because it preserves non-covalent interactions. Matrix-assisted laser desorption/ionization (MALDI) represents an important alternative to ESI because it is more tolerant to salts and detergents (e.g. necessary in the case of membrane complex analyses). Prior to MALDI-MS, the subunits should be crosslinked (XLed). Moreover, crosslinking (XLing) is useful when constraint distances are determined to obtain low-resolution structural information. Here we report a novel XLing approach to study protein complexes with MALDI-MS. We investigated two tetramers (i.e. alcohol dehydrogenase and aldolase) larger than 140 kDa at two pH values (7.2 and 8.0). We tested two different crosslinkers (XLers) (i.e. BS(3) and glutaraldehyde), used separately or in combination. We utilized gentle agitation and ultracentrifugation. Our data shows that the pH influenced the XLing when using a single XLer. Combining two XLers was demonstrated to be more efficient than using a reagent alone. In particular, the combination determined a higher degree of XLing and lower mass shift. This could suggest a ranking in target amino acid availability. First residues at specific distances are linked by BS(3) , then glutaraldehyde binds residues that are still available at larger distances. Ultracentrifugation and gentle agitation both provide similar degrees of XLing, but the former method determined a lower mass increment resulting from redundant XLing. To conclude, we present an efficient dual XLing approach for determining mass and stoichiometry of protein assemblies.

Optimization of N-hydroxysuccinimide ester coupling with aminoallyl-modified RNA for fluorescent labeling

“…However, restricting by experimental cost, the concentrations of aa-RNA was just increased to 0.3 mM, higher concentration of aa-RNA are suggested for users in future studies, which may further enhance the reaction efficiency and reduce the cost. Unlike most of protocols for DNA or protein labeling [ 24 , 27 , 28 ], this work demonstrated that the optimal concentration of DMSO is 45–55% (vol/vol). Suggesting that sufficient DMSO was required to achieve efficient labeling of aa-RNA, and the concentration of DMSO should be kept at 45–55% (vol/vol) while avoiding high (>70%) or low (<30%) concentration of DMSO.…”

“…However, restricting by experimental cost, the concentrations of aa-RNA was just increased to 0.3 mM, higher concentration of aa-RNA are suggested for users in future studies, which may further enhance the reaction efficiency and reduce the cost. Unlike most of protocols for DNA or protein labeling [24,27,28], this work demonstrated that the optimal concentration of DMSO is 45-55% (vol/vol).…”

Tris(hydroxymethyl)aminomethane Compatibility with N-Hydroxysuccinimide Ester Chemistry: Biotinylation of Peptides and Proteins in TRIS Buffer

“…In current protocols, it is strongly recommended to remove this chemical before the reaction with NHS esters, since it involves a primary amino functional group and is usually present in the 100 mM concentration range. Moreover, TRIS has been proposed as a reagent to block excess NHS esters. , …”

“…Moreover, TRIS has been proposed as a reagent to block excess NHS esters. 1,8 Here, we inadvertently biotinylated a nanobody which is specific for green-fluorescent protein (GFP) 9 between a nanobody biotinylated in the presence or absence of 100 mM TRIS (Figure 1). Based on these results, we hypothesized that the pH might have an impact on the biotinylation reaction and that a higher or lower pH might lead to a preferred reaction with the amino groups of the antibody compared to the function in TRIS.…”