A neuroproteomic approach to targeting neuropeptides in the brain

Abstract

A novel universal neuropeptide display approach in the mass range of 300-5000 Da was developed to complement two-dimensional gel electrophoresis in the analysis of peptides and small proteins from brain tissue samples. For the analysis of neuropeptides we utilized on-line nanoscale capillary reversed phase liquid chromatography and electrospray ionization quadrupole-time of flight mass spectrometry. The method was employed for the analysis of a large number of peptides from three specific rat brain regions. Approximately 1500 peptides from each brain region were detected in the same analysis. Several of these peptides were sequenced using collision-induced dissociation and identified by database search tools. In addition, a method for comparing peptide elution profiles between samples was developed, to provide two- and three-dimensional computer graphics of the profiles and to pinpoint differences for statistical measurements. Among the characterized peptides were fragments from proteins such as hemoglobin, alpha-synuclein, stathmin, cyclophilin, actin, NADH dehydrogenase, cytochrome c oxidase and prosomatostatin, as well as the bioactive neuropeptides W-hemorphin-4, and LW-hemorphin-7. The present study showed that the combination of nanoscale reversed phase liquid chromatography and high-resolution tandem mass spectrometry provides a novel and powerful approach to investigate a large number of peptides and protein fragments in the brain.

Heat Stabilization of the Tissue Proteome: A New Technology for Improved Proteomics

“…Mice in a fourth sample group (n ) 4) were killed by focused microwave irradiation (1.4 s at 4.5-5 kW, Muromachi Kikai, Tokyo, Japan) which denature the proteins before dissection and homogenization. A microtip ultrasonicator (Vibra-Cell, Sonics & Materials) was used to homogenize the tissue in 5× the sample weight of 0.25% HAc extraction solution containing 40 mM of isotopically labeled stathmin (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20), met-enkephalin, neurotensin, substance P (1-11), and substance P (1-7) as internal standards. The crude extract was centrifuged at 20 000g for 30 min at 4°C to pellet cell debris and ultrafiltered using a 10 kDa centrifugal cutoff filter (Microcon YM-10, Millipore) to isolate the peptide content.…”

Neuropeptidomics: MS Applied to the Discovery of Novel Peptides from the Brain

“…However, early contributions to this field in terms of novel peptide identifications and characterizations were made from experiments on invertebrates, such as locust, fruit fly, and flesh fly, even before the term peptidomics was invented (21,22). Peptidomics methodologies are generally based on separating complex endogenous peptide mixtures by LC-usually nanoscale capillary reversed-phase LC (nanoLC; 23,24), multistep chromatographic approaches (25,26), or gel-or liquid-based isoelectric focusing (27 )-combined with MS to identify peptides.…”

“…Hence, neuropeptidomics is the technological approach for detailed analyses of endogenous peptides from the brain (5,23,(30)(31)(32)(33)(34). The levels of peptides in the brain reflect certain information about physiological status; this information can be revealed when MS is used to generate broad profiles of the dynamic neuropeptide pattern.…”

“…This qualitative and quantitative procedure has been termed "differential peptide display" (4,23,35). The MS approach, with either ESI (36) or MALDI (37 ), permits sensitive, simultaneous detection of peptide changes in complex mixtures of hundreds of different peptides.…”

Comprehensive Peptidome Analysis of Mouse Livers by Size Exclusion Chromatography Prefractionation and NanoLC−MS/MS Identification

“…14,15 The endogenous peptides play crucial roles in the respiratory, cardiovascular, endocrine, inflammatory, and nervous systems. 11,16 The discovery of novel neuropeptides has been extensively studied, and some databases have been established for the endogenous peptides. 12,[17][18][19] The degraded fragments of proteins are generated by the proteolytic enzymes in the body, 20 which can be considered as the metabolic products of proteins.…”