All-in-One Pseudo-MS³ Method for the Analysis of Gas-Phase Cleavable Protein Crosslinking Reactions

Crosslinking mass spectrometry (XL-MS) supports structure analysis of individual proteins and highly complex whole-cell interactomes. The identification of crosslinked peptides from enzymatic digests remains challenging, especially at the cell level. Empirical methods that use gas-phase cleavable crosslinkers can simplify the identification process by enabling an MS3-based strategy that turns crosslink identification into a simpler problem of detecting two separable peptides. However, the method is limited to select instrument platforms and is challenged by duty cycle constraints. Here, we revisit a pseudo-MS3 concept that incorporates in-source fragmentation, where a fast switch between gentle high-transmission source conditions and harsher in-source fragmentation settings liberates peptides for standard MS2-based peptide identification. We present an all-in-one method where retention time matches between the crosslink precursor and the liberated peptides establish linkage, and MS2 sequencing identifies the source-liberated peptides. We demonstrate that DC4, a very labile cleavable crosslinker, generates high-intensity peptides in-source. Crosslinks can be identified from these liberated peptides, as they are chromatographically well-resolved from monolinks. Using bovine serum albumin (BSA) as a crosslinking test case, we detect 27% more crosslinks with pseudo-MS3 over a best-in-class MS3 method. While performance is slightly lower for whole-cell lysates (generating two-thirds of the identifications of a standard method), we find that 60% of these hits are unique, highlighting the complementarity of the method

All-in-One Pseudo-MS³ Method for the Analysis of Gas-Phase Cleavable Protein Crosslinking Reactions

Crosslinking mass spectrometry (XL-MS) supports structure analysis of individual proteins and highly complex whole-cell interactomes. The identification of crosslinked peptides from enzymatic digests remains challenging, especially at the cell level. Empirical methods that use gas-phase cleavable crosslinkers can simplify the identification process by enabling an MS3-based strategy that turns crosslink identification into a simpler problem of detecting two separable peptides. However, the method is limited to select instrument platforms and is challenged by duty cycle constraints. Here, we revisit a pseudo-MS3 concept that incorporates in-source fragmentation, where a fast switch between gentle high-transmission source conditions and harsher in-source fragmentation settings liberates peptides for standard MS2-based peptide identification. We present an all-in-one method where retention time matches between the crosslink precursor and the liberated peptides establish linkage, and MS2 sequencing identifies the source-liberated peptides. We demonstrate that DC4, a very labile cleavable crosslinker, generates high-intensity peptides in-source. Crosslinks can be identified from these liberated peptides, as they are chromatographically well-resolved from monolinks. Using bovine serum albumin (BSA) as a crosslinking test case, we detect 27% more crosslinks with pseudo-MS3 over a best-in-class MS3 method. While performance is slightly lower for whole-cell lysates (generating two-thirds of the identifications of a standard method), we find that 60% of these hits are unique, highlighting the complementarity of the method

Lactoferrin binding protein B – a bi-functional bacterial receptor protein

<div><p>Lactoferrin binding protein B (LbpB) is a bi-lobed outer membrane-bound lipoprotein that comprises part of the lactoferrin (Lf) receptor complex in <i>Neisseria meningitidis</i> and other Gram-negative pathogens. Recent studies have demonstrated that LbpB plays a role in protecting the bacteria from cationic antimicrobial peptides due to large regions rich in anionic residues in the C-terminal lobe. Relative to its homolog, transferrin-binding protein B (TbpB), there currently is little evidence for its role in iron acquisition and relatively little structural and biophysical information on its interaction with Lf. In this study, a combination of crosslinking and deuterium exchange coupled to mass spectrometry, information-driven computational docking, bio-layer interferometry, and site-directed mutagenesis was used to probe LbpB:hLf complexes. The formation of a 1:1 complex of iron-loaded Lf and LbpB involves an interaction between the Lf C-lobe and LbpB N-lobe, comparable to TbpB, consistent with a potential role in iron acquisition. The Lf N-lobe is also capable of binding to negatively charged regions of the LbpB C-lobe and possibly other sites such that a variety of higher order complexes are formed. Our results are consistent with LbpB serving dual roles focused primarily on iron acquisition when exposed to limited levels of iron-loaded Lf on the mucosal surface and effectively binding apo Lf when exposed to high levels at sites of inflammation.</p></div

LbpB mutants.

<p>LbpB mutants.</p

Specificity of LbpB and TbpB for iron-loaded glycoprotein.

<p>(A) Competitive solid-phase binding assay of TbpB with apo/holo hTf and LbpB with apo/holo hLf. Recombinant MBP-TbpB (top two rows) and MBP-LbpB (bottom two rows) were applied to nitrocellulose paper, the paper blocked and then incubated with apo- or holo- glycoprotein overnight in a ¼ serially diluted fashion (A, 20nM; B, 5nM; C, 1.25nM; D, 0.31nM; E, 0.07nM; F, 0.01nM; G, 4.88 × 10^-3nM; H, 0nM). Iron-loaded HRP-conjugated glycoprotein (HRP-hTf or HRP-hLf) was then introduced into the binding mixture. Presence of a dot represents the displacement of any protein bound to TbpB or LbpB by the HRP-conjugate at the given concentration. (B) SDS-PAGE/affinity capture representing receptor protein (MBP-TbpB, 122kDa; or MBP-LbpB, 122kDa) captured by Sepharose resins conjugated to their cognate apo- or holo-glycoprotein (hTf-r, hLf-r).</p

Proposed functions of LbpB.

<p>(LEFT) LbpB may be involved in the iron-acquisition pathway. At low concentrations of holo-hLf, LbpB may use its LbpB-N binding mode to preferentially bind iron-loaded lactoferrin and shuttle it to LbpA, forming a ternary complex and hijacking the iron. (RIGHT) Cleavage of LbpB from the membrane may be dependent on the presence of high levels of hLf in the extracellular milieu or simply a constitutive property of <i>N</i>. <i>meningitidis</i> cells in the NalP phase-variable ON-state. The release of LbpB from the membrane is done in an effort to sequester lactoferricin, antibodies, and possibly form large lattices of hLf as to prevent proteolytical processing into its derivative cationic antimicrobial peptides.</p

Receptor lobe binding contributions in TbpB and LbpB.

<p>Cartoon representations of each recombinant LbpB protein are displayed beside their respective BLI steady-state binding curve from binding hLf. (A) Intact LbpB, K_{D app} = 72.8 ± 3.24nM. (B) LbpB-N lobe, K_{D app} = 126 ± 48nM. (C) LbpB-C lobe K_{D app} = 279 ± 15nM. C-lobe Hill slope was calculated to be 1.98 ± 0.13 implying positive cooperativity. (D) Intact-lgsm, K_{D app} = 140 ±82.4nM (E). LbpB-C lobe-lgsm had no observed binding.</p