Multi-Faceted Mass Spectrometric Investigation of Neuropeptides in Callinectes sapidus

Summary

Mass spectrometric characterization of neuropeptides provides sequence, quantitation, and localization information. This optimized workflow is not only useful for neuropeptide studies, but also other endogenous peptides. The protocols provided here describe sample preparation, MS acquisition, MS analysis, and database generation of neuropeptides using LC-ESI-MS, MALDI-MS spotting, and MALDI-MS imaging.

Abstract

Neuropeptides are signaling molecules that regulate almost all physiological and behavioral processes, such as development, reproduction, food intake, and response to external stressors. Yet, the biochemical mechanisms and full complement of neuropeptides and their functional roles remain poorly understood. Characterization of these endogenous peptides is hindered by the immense diversity within this class of signaling molecules. Additionally, neuropeptides are bioactive at concentrations 100x - 1000x lower than that of neurotransmitters and are prone to enzymatic degradation after synaptic release. Mass spectrometry (MS) is a highly sensitive analytical tool that can identify, quantify, and localize analytes without comprehensive a priori knowledge. It is well-suited for globally profiling neuropeptides and aiding in the discovery of novel peptides. Due to the low abundance and high chemical diversity of this class of peptides, several sample preparation methods, MS acquisition parameters, and data analysis strategies have been adapted from proteomics techniques to allow optimal neuropeptide characterization. Here, methods are described for isolating neuropeptides from complex biological tissues for sequence characterization, quantitation, and localization using liquid chromatography (LC)-MS and matrix-assisted laser desorption/ionization (MALDI)-MS. A protocol for preparing a neuropeptide database from the blue crab, Callinectes sapidus, an organism without comprehensive genomic information, is included. These workflows can be adapted to study other classes of endogenous peptides in different species using a variety of instruments.

Introduction

The nervous system is complex and requires a network of neurons to transmit signals throughout an organism. The nervous system coordinates sensory information and biological response. The intricate and convoluted interactions involved in signal transmission require many different signaling molecules such as neurotransmitters, steroids, and neuropeptides. As neuropeptides are the most diverse and potent signaling molecules that play key roles in activating physiological responses to stress and other stimuli, it is of interest to determine their specific role in these physiological processes. Neuropeptide function is related to their amino acid structure, which determin....

Protocol

All tissue sampling described was performed in compliance with the University of Wisconsin-Madison guidelines.

1. LC-ESI-MS analysis of neuropeptides

1. Neuropeptide extraction and desalting
   1. Prior to tissue acquisition, prepare acidified methanol (acMeOH) (90:9:1 MeOH:water:acetic acid) as described in¹⁶.
   2. Collect brain tissue from the crustacean¹⁷ and use forceps to immediately place one tissue each in a 0.6 mL tube containing 20 µL of acMeOH.
      NOTE: Tissue dissection protocols vary greatly for different animals and different tissue ....

Results

The workflow for sample preparation and MS analysis is depicted in Figure 1. After the dissection of neuronal tissue, homogenization, extraction, and desalting are performed to purify neuropeptide samples. If isotopic label-based quantification is desired, samples are then labeled and desalted once again. The resulting sample is analyzed through LC-MS/MS for neuropeptide identification and quantification.

Neuropeptides identified through the proteomics software sh.......

Discussion

The accurate identification, quantification, and localization of neuropeptides and endogenous peptides found in the nervous system are crucial toward understanding their function^23,24. Mass spectrometry is a powerful technique that can allow all of this to be accomplished, even in organisms without a fully sequenced genome. The ability of this protocol to detect, quantify, and localize neuropeptides from tissue collected from C. sapidus through a combina.......

Materials

Name	Company	Catalog Number	Comments
Chemicals, Reagents, and Consumables
2,5-Dihydroxybenzoic acid (DHB) matrix	Supelco	39319
Acetic acid	Fisher Chemical	A38S-500
Acetonitrile Optima LC/MS grade	Fisher Chemical	A955-500
Ammonium bicarbonate	Sigma-Aldrich	9830
Borane pyridine	Sigma-Aldrich	179752
Bruker peptide calibration mix	Bruker Daltonics	NC9846988
Capillary	Polymicro	1068150019	to make nanoflow column (75 µm inner diameter x 360 µm outer diameter)
Cryostat cup	Sigma-Aldrich	E6032	any cup or mold should work
Microcentrifuge Tubes	Eppendorf	30108434
Formaldehyde	Sigma-Aldrich	252549
Formaldehyde - D2	Sigma-Aldrich	492620
Formic acid Optima LC/MS grade	Fisher Chemical	A117-50
Gelatin	Difco	214340	place in 37 °C water bath to melt
Hydrophobic barrier pen	Vector Labs	15553953
Indium tin oxide (ITO)-coated glass slides	Delta Technologies	CB-90IN-S107	25 mm x 75 mm x 0.8 mm (width x length x thickness)
LC-MS vials	Thermo	TFMSCERT5000-30LVW
Methanol Optima LC/MS Grade	Fisher Chemical	A456-500
Parafilm	Sigma-Aldrich	P7793	Hydrophobic film
pH-Indicator strips	Supelco	109450
Red phosphorus clusters	Sigma-Aldrich	343242
Reversed phase C18 material	Waters	186002350	manually packed into nanoflow column
Wite-out pen	BIC	150810
ZipTip	Millipore	Z720070
Instruments and Tools
Automatic matrix sprayer system- M5	HTX Technologies, LLC
Centrifuge - 5424 R	Eppendorf	05-401-205
Cryostat- HM 550	Thermo Fisher Scientific	956564A
Desiccant	Drierite	2088701
Forceps	WPI	501764
MALDI stainless steel target plate	Bruker Daltonics	8280781
Pipet-Lite XLS	Rainin	17014391	200 µL
Q Exactive Plus Hybrid Quadrupole-Orbitrap	Thermo Fisher Scientific	IQLAAEGAAPFALGMBDK
RapifleX MALDI-TOF/TOF	Bruker Daltonics
SpeedVac - SVC100	Savant	SVC-100D
Ultrasonic Cleaner	Bransonic	2510R-MTH	for sonication
Ultrasonic homogenizer	Fisher Scientific	FB120110	FB120 Sonic Dismembrator with CL-18 Probe
Vaccum pump- Alcatel 2008 A	Ideal Vacuum Products	P10976	ultimate pressure = 1 x 10-4 Torr
Vortex Mixer	Corning	6775
Water bath (37C) - Isotemp 110	Fisher Scientific	15-460-10
Data Analysis Software
Expasy	https://web.expasy.org/translate/
FlexAnalysis	Bruker Daltonics
FlexControl	Bruker Daltonics
FlexImaging	Bruker Daltonics
PEAKS Studio	Bioinformatics Solutions, Inc.
SCiLS Lab	https://scils.de/
SignalP 5.0	https://services.healthtech.dtu.dk/service.php?SignalP-5.0
tBLASTn	http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=tblastn&BLAST_ PROGRAMS=tblastn&PAGE_ TYPE=BlastSearch&SHOW_ DEFAULTS=on&LINK_LOC =blasthome