Analytic framework for peptidomics applied to large-scale neuropeptide identification

Abstract

Large-scale mass spectrometry-based peptidomics for drug discovery is relatively unexplored because of challenges in peptide degradation and identification following tissue extraction. Here we present a streamlined analytical pipeline for large-scale peptidomics. We developed an optimized sample preparation protocol to achieve fast, reproducible and effective extraction of endogenous peptides from sub-dissected organs such as the brain, while diminishing unspecific protease activity. Each peptidome sample was analysed by high-resolution tandem mass spectrometry and the resulting data set was integrated with publically available databases. We developed and applied an algorithm that reduces the peptide complexity for identification of biologically relevant peptides. The developed pipeline was applied to rat hypothalamus and identifies thousands of neuropeptides and their post-translational modifications, which is combined in a resource format for visualization, qualitative and quantitative analyses.

Improved identification and quantitation of mature endogenous peptides in the rodent hypothalamus using a rapid conductive sample heating system

“…Recently, Secher et al 42 reported on an analytical framework for peptide characterization using the longest peptide variant (LPV) method, where peptides were assembled into LPVs to account for the sequential degradation ladder sequences due to non-specific enzymatic activity. Using this approach, 14,416 unique peptide sequences were grouped into 2,835 LPVs, of which 356 were derived from prohormone precursors.…”

“…Though the mass spectrometric platforms used in both studies are different, we posit that the principal factor contributing to this dissimilarity could be the difference in the peptide extraction method used. Secher et al 42 used urea, which is a preferred choice for protein extraction as it helps dissolve the protein, but may not be optimal for peptides, especially as peptides are smaller and more easily dissolvable compared to proteins. Moreover, the otherwise insoluble proteins would now be soluble, with a potential to degrade during sample processing, and thus can mask some of the trace-level endogenous peptides.…”

“…20,21,25 The ST1 benchtop instrument heats fresh and frozen samples quickly and uniformly by rapid conductive heating through metallic blocks pressed against the sample. This technique has been reported to successfully retain the endogenous proteome and peptidome close to the in vivo state, as assessed in previous studies 33,40–42 including analysis of the mature neuropeptide complement in crustaceans 39 and snap-frozen mammalian tissues. 38 …”

“…25 The distinction is important as full-length peptides are produced, accumulated, and released from cells in-vivo , 1–3 and are more likely to be biologically active, whereas randomly truncated forms often observed in the MS measurements could be sampling artifacts caused by the loss of the sample cellular integrity and release of lysosomal proteases, or by chemical degradation under the extreme pH conditions typical for peptide extract preparation. Ladder sequence truncation, as evidenced in the supplemental data set by Secher et al 42 for example, is a well-known form of peptide chemical degradation under extreme pH that had been used for peptide sequencing before the advent of tandem MS (MS/MS)-capable instrumentation. 43 …”

Human POMC processing in vitro and in vivo revealed by quantitative peptidomics

“…Other differences could be explained by the relative immaturity of hPSC-derived hypothalamic neurons at the time points we studied, resulting in incomplete pro-peptide processing or expression at levels too low to be automatically detectable using the automated peptide identification pipeline we employed. The use of more sensitive LC-MS/MS instruments or the analysis of raw peptidomic data using new discovery tools 69 may reveal further biologically important peptides. We found that exposure of hPSC-derived hypothalamic neurons to leptin significantly increased the concentration of both a-MSH and b-MSH, and that these peptides were robustly secreted from upon stimulation.…”