A crustacean neuropeptide spectral library for data‐independent acquisition (DIA) mass spectrometry applications

Abstract

Neuropeptides have tremendous potential for application in modern medicine, including utility as biomarkers and therapeutics. To overcome the inherent challenges associated with neuropeptide identification and characterization, data‐independent acquisition (DIA) is a fitting mass spectrometry (MS) method of choice to achieve sensitive and accurate analysis. It is advantageous for preliminary neuropeptidomic studies to occur in less complex organisms, with crustacean models serving as a popular choice due to their relatively simple nervous system. With spectral libraries serving as a means to interpret DIA‐MS output spectra, and Cancer borealis as a model of choice for neuropeptide analysis, we performed the first spectral library mapping of crustacean neuropeptides. Leveraging pre‐existing data‐dependent acquisition (DDA) spectra, a spectral library was built using PEAKS Online. The library is comprised of 333 unique neuropeptides. The identification results obtained through the use of this spectral library were compared with those achieved through library‐free analysis of crustacean brain, pericardial organs (PO), and thoracic ganglia (TG) tissues. A statistically significant increase (Student's t‐test, P value < 0.05) in the number of identifications achieved from the TG data was observed in the spectral library results. Furthermore, in each of the tissues, a distinctly different set of identifications was found in the library search compared to the library‐free search. This work highlights the necessity for the use of spectral libraries in neuropeptide analysis, illustrating the advantage of spectral libraries for interpreting DIA spectra in a reproducible manner with greater neuropeptidomic depth.

EndoGenius: Optimized Neuropeptide Identification from Mass Spectrometry Datasets

“…While the underlying mechanistic reason for this significance was not probed, it can be speculated that this is due to the discrepancy in ionization efficiency, kinetic stability, and subsequent fragment ion abundance between b- and y-type fragment ions. In fact, with nontryptic, doubly charged peptides, a higher abundance of b-type fragment ions has been noted previously, and it is perhaps a reasonable hypothesis given the high propensity for neuropeptide precursors to appear at a +2 charge state . Interestingly, a handful of significant factors had widely diverging optimal values across tissue types (Figure S4).…”

“…Neuropeptides, essential signaling molecules originating from neurons or endocrine cells, serve as a resource for obtaining dynamic information on neuronal processes . To date, neuropeptides have provided valuable insight into biological disorders including Alzheimer’s disease , and obesity. − Mass spectrometry (MS) is revered for its high accuracy and sensitivity and has been utilized in many neuropeptide studies to date. − With regard to processing MS data and subsequently obtaining peptide–spectrum matches (PSMs), database searching is a routine method, which compares the theoretical spectrum of a peptide with an experimental spectrum to make identifications. The area of database searching has seen tremendous growth in recent decades, with many software packages available for use including MSFragger, MaxQuant, PEAKS, Comet, and MetaMorpheus, each of which were designed for application in proteomics investigations.…”