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Simple and Fast Maximally Deuterated Control (maxD) preparation for HDX MS Experiments

ABSTRACT: During the analysis steps of hydrogen deuterium exchange (HDX) mass spectrometry (MS) there is an unavoidable loss of deuterons, or back-exchange. Understanding back-exchange is necessary to correct for loss during analysis, to calculate the absolute amount of exchange, and to ensure that deuterium recovery is as high as possible during LC-MS. Back-exchange can be measured and corrected for using a maximally deuterated species (here called maxD) in which the protein is deuterated at all backbone amide positions and analyzed with the same buffer components, %D2O, quenching conditions, and LC-MS parameters used during the analysis of other labeled samples. Here we describe a robust and broadly applicable protocol, using denaturation followed by deuteration, to prepare a maxD control sample in ~40 minutes. The protocol was evaluated with a number of proteins that varied in both size and folded structure. The relative fractional uptake and level of back-exchange with this protocol were both equivalent to those obtained with a previous protocol that requires much more time or one requiring isolation of peptic peptides prior to deuteration. Placing strong denaturation first in the protocol allowed maximum deuteration in a short time (~10 mins) with equal or more deuteration found in other methods. The absence of high temperatures and low pH during the deuteration step limited protein aggregation. This high-performance, fast, and easy to perform protocol should enhance routine preparation of maxD controls for both beginners and for challenging proteins.

INSTRUMENT(S): Synapt MS

ORGANISM(S): Equus Caballus (horse) Homo Sapiens (human) Bos Taurus (bovine)

SUBMITTER: John R. Engen

LAB HEAD: John R. Engen

PROVIDER: PXD032924 | Pride | 2022-07-08

REPOSITORIES: Pride

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Project description:Collagen deposition is a key process during idiopathic pulmonary fibrosis (IPF); however, little is known about the dynamics of collagen formation during disease development. Tissue samples of early stages of human disease are not readily available and it is difficult to identify changes in collagen content, since standard collagen analysis does not distinguish between 'old' and 'new' collagen. Therefore, the current study aimed to (i) investigate the dynamics of new collagen formation in mice using bleomycin-induced lung fibrosis in which newly synthesized collagen was labelled with deuterated water and (ii) use this information to identify genes and processes correlated to new collagen formation from gene expression analysis. Lung fibrosis was induced in female C57BL/6 mice by bleomycin instillation and sacrificed. Animals were sacrificed at 1 to 5 weeks after fibrosis induction. Collagen synthesized during the week before sacrifice was labelled with deuterium by providing mice with deuterated drinking water. After sacrifice, lung tissue was collected for microarray analysis, determination of new collagen formation, and histology. Deuterated water labelling showed a strong increase in new collagen formation already during the first week after fibrosis induction and a complete return to baseline at five weeks. Correlation of new collagen formation data with gene expression data revealed fibrosis specific processes, of which proliferation was an unexpected one. This was confirmed by measuring cell proliferation and collagen synthesis simultaneously using deuterated water incorporation. Furthermore, new collagen formation strongly correlated with gene expression of e.g. elastin, tenascin C, MMP-14, lysyl oxidase, and type V collagen. These data demonstrate, using a novel combination of technologies, that proliferation and extracellular matrix production are correlated to the core process of fibrosis, i.e. the formation of new collagen. In addition, it identified genes directly correlated to fibrosis, thus providing more insight into the aetiology of IPF. Total RNA was obtained from mouse lungs at timepoint 0 as a control (n = 7) or timepoints 1 (n = 7), 2 (n = 6), 3 (n = 6), 4 (n = 6) or 5 (n = 6) weeks after bleomycin-instillation to induce lung fibrosis.

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Simple and Fast Maximally Deuterated Control (maxD) preparation for HDX MS Experiments

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