Excess Salt Removal with Matrix Rinsing: Direct Peptide Profiling of Neurons from Marine Invertebrates Using Matrix-assisted Laser Desorption/Ionization Time-of-flight Mass Spectrometry

Abstract

Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI/TOF‐MS) is a viable technique for the examination of biological environments. Clearly, sample preparation plays a pivotal role in the ability to obtain mass spectra from samples as complex as biological cells. The physiological salt concentrations associated with neurons from marine specimens interfere with MALDI analysis. A unique and simple rinsing procedure allows cellular clusters, individual neurons and connective tissues to be directly assayed for peptides with minimal sample handling. Isolated cells and tissues, including egg‐laying hormone‐releasing cells, from the central nervous systems of the model marine molluscs Aplysia californicaand Pleurobranchaea californicaare used to demonstrate the salt removal method. In addition to facilitating sample ionization, the MALDI matrix 2,5‐dihydroxybenzoic acid serves to (i) aid in microdissections by stabilizing cell membranes, (ii) deactivate endogenous proteolytic enzymes and (iii) reduce high salt concentrations in order to improve spectral quality. Representative MALDI mass spectra are presented which indicate the presence of several neuroactive peptides previously characterized by conventional biochemical methods. More than ten individual peptides can be detected in a single cell. In spite of the chemically complex sample, the mass spectra are surprisingly free of extraneous peaks. Furthermore, both mass resolution and mass accuracy are similar to those encountered with more common MALDI samples and protocols.

Identification and Characterization of the Feeding Circuit-Activating Peptides, a Novel Neuropeptide Family ofAplysia

“…Mass spectrometr y. M ALDI-TOF MS of neurons was performed as described previously (Garden et al, 1996;Fujisawa et al, 1999;Furukawa et al, 2001). Analysis of the CBC s was done on short sections (Ͻ1 mm) of the desheathed cut ends of CBC s after varying periods of incubation in Leibovitz's L -15 media (Life Technologies).…”

Gold nanoparticles as assisted matrices for the detection of biomolecules in a high-salt solution through laser desorption/ionization mass spectrometry

“…Expectedly, in the case of 2,5-DHB, the signal intensities of protonated steroids gradually decreased with increasing concentration of NaCl (Figure 1a). This is primarily attributed to the high concentration of NaCl, which interferes with matrix crystal formation, resulting in signal suppression [26]. Another reason is that the presence of NaCl in a sample solution can cause the formation of the sodium adduct ions during the desorption/ionization process, leading to the low production of protonated ions [27].…”

Tissue Proteomics for the Next Decade? Towards a Molecular Dimension in Histology

“…Soon after, in the early 1990s, authors benefited from the fact that samples for MALDI need to be solid to study mollusk neuropeptides processing directly on animals sections ( Jimenez et al, 1994). The method was then extended to other models (Garden et al, 1996;Stahl et al, 1997;van Strien et al, 1996). In 1997, Caprioli proposed an improvement of the method consisting in its automation in order to acquire spectra from areas of the samples.…”