Direct visualisation of peptide hormones in cultured pancreatic islet alpha‐ and beta‐cells by intact‐cell mass spectrometry

Abstract

The application of intact-cell mass spectrometry (ICM) by matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry to achieve direct protein-profiling of bacterial species is now well established. However, this methodology has not to our knowledge been applied to the analysis of mammalian cells in routine culture. Here, we describe a novel application of ICM by which we have identified proteins in intact cells from two lines representative of pancreatic islet alpha- and beta-cells. Adherent alphaTC1 clone 9 and betaTC6 F7 cells were harvested into phosphate-buffered saline (PBS) using enzyme-free dissociation buffer before 1 microL of cell suspension was spotted onto MALDI plates. Cells were overlaid with sinapinic acid then washed with pure water before application of a final coat of sinapinic acid. Data in the 2000-20,000 m/z range were acquired in linear mode on a Voyager DE-Pro mass spectrometer. The proteins which ionised were composed in large part of peptide hormones (e.g. insulin and glucagon) known to be packaged into the secretory granules of the beta- and alpha-cells respectively. However, in addition to visualising the peptides expected to be associated with these cells, a mass consistent with oxyntomodulin was identified in the cultured alpha-cells, a finding not previously reported to our knowledge. In summary, this paper describes, for the first time, a rapid and direct method useful for identifying secretory products in intact endocrine cells.

Processing of proglucagon to GLP-1 in pancreatic α-cells: is this a paracrine mechanism enabling GLP-1 to act on β-cells?

“…Although glucagon is the major product of the proglucagon gene in the aTC1-6 cell line, the data presented shows that PC1 is expressed and that GLP-1 is produced and detectable in culture medium after only 30 min incubation. This is substantiated by the finding of GLP-1 along with glucagon and oxyntomodulin, using the more qualitative approach of intactcell mass spectrometry in aTC1-9 cells (Buchanan et al 2007). In addition, GLP-1 is present in isolated human pancreatic islets cultured for 24 h, raising the possibility that upregulation of this mechanism could provide GLP-1 for paracrine effects.…”

Aluminium foil as a single-use substrate for MALDI-MS fingerprinting of different melanoma cell lines

“…In this case, cells can be either grown directly on a MALDI target plate 19 or collected by centrifugation after culturing in a classical Petri dish. 11,[20][21][22][23] The latter allows cell pellets to be either transferred directly to the target plate, where they are dried and covered with a matrix solution, 22,24 or mixed with a matrix solution prior to the transfer. 20,21,23,25,26 As a result, instead of individual protein peaks, a number of signals representing the MS fingerprint characteristic for a specific cell type or physiological state can be obtained.…”

Whole-cell MALDI-TOF Mass Spectrometry is an Accurate and Rapid Method to Analyze Different Modes of Macrophage Activation

“…Many applications may be derived from this technique: (1) identify the main cell types from a mixed sample [9][10][11] ; (2) assess the viability of cell cultures over time (including quality control industrial applications) 12 ; (3) monitor activation states of a single cell type 13 ; (4) assess the malignant transformation of a clinical sample 14,15 .…”