Liquid chromatography/mass spectrometry based detection and semi‐quantitative analysis of INSL5 in human and murine tissues

Abstract

RationaleInsulin‐like peptide 5 (INSL5) is a hormone produced by enteroendocrine L‐cells in the colon that has recently been implicated in the control of metabolic homeostasis. However, research into its physiology has been hindered by the reported unreliability of commercially available immunoassays and additional detection assays would benefit this emerging field.MethodsPeptides from purified murine L‐cells and homogenates from both human and mouse colonic tissues were extracted by precipitating larger proteins with acetonitrile. Untargeted liquid chromatography/tandem mass spectrometry (LC/MS/MS) analyses, followed by database searching, were used to detect and identify various INSL5 gene derived peptides and characterise their precise sequence. A similar approach was developed to quantify INSL5 levels in primary intestinal culture supernatants after purification and concentration by solid‐phase extraction.ResultsMass spectral analysis of purified enteroendocrine cells and tissue homogenates identified the exact sequence of A and B chains of INSL5 endogenously expressed in L‐cells. Differences in the endogenously processed peptide and the Swissprot database entry were observed for murine INSL5, whereas the human sequence matched previous predictions from heterologous expression experiments. INSL5 was detected in the supernatant of human and mouse primary colonic cultures and concentrations increased after treatment with a known L‐cell stimulus.ConclusionsThe first LC/MS/MS‐based method capable of the detection and semi‐quantitative analysis of endogenous INSL5 using MS‐based techniques has been demonstrated. The methodology will enable the identification of stimulants for INSL5 secretion from murine and human primary colonic epithelial cultures.

Comparison of human and murine enteroendocrine cells by transcriptomic and peptidomic profiling

“…Samples were extracted using a Waters HLB μElution solid-phase extraction (SPE) plate (Waters, MA, USA) after being resuspended in 500μL 0.1% v/v formic acid in water as described previously and analysed after reduction/alkylation(36). Human homogenates and mouse homogenates for interspecies comparison were analysed using nano-flow based separation and electrospray approaches on a Thermo Fisher Ultimate 3000 nano LC system coupled to a Q Exactive Plus Orbitrap mass spectrometer (ThermoScientific).…”

Human POMC processing in vitro and in vivo revealed by quantitative peptidomics

“…We therefore wondered what the relative concentrations of POMC-derived peptides relevant for human energy homeostasis might be in vitro and in vivo. To address this question, we developed quantitative assays 53 for α-MSH(1-13), d-α-MSH(1-13), β-MSH(1-18) and β-EP(1-31) (Fig. 4A).…”

“…EDTA was then removed and cells were directly lysed by vigorously pipetting cells with 80% acetonitrile (ACN, Pierce Cat# 51101), an organic solvent that is routinely used to precipitate larger proteins and protein complexes 53,73 . This lysate was then transferred to low protein-binding microcentrifuge tube (Sigma, Cat# Z666505), vortexed for 1 minute, and pelleted for 15 minutes at 12,000 g at 4˚C.…”

“…Peptide extracts were run using nano---flow---based separation and electrospray approaches on a Thermo Fisher Ultimate 3000 nano---LC system coupled to a Q Exactive Plus Orbitrap mass spectrometer (ThermoScientific, San Jose, USA) as described previously 53 .…”

Mass spectrometric characterisation of the circulating peptidome following oral glucose ingestion in control and gastrectomised patients

“…In our view a better approach for analysing peptides by mass spectrometry in an untargeted fashion is to avoid enzymatic digestion 12 . We previously reported such an approach for plasma 13 from patients with endocrine tumours and for sorted cells 14 and tissue extracts from human brain 15 and intestines 16 . In this study, we compared changes in the plasma peptidome during an oral glucose tolerance test (OGTT) in subjects after gastrectomy surgery (a procedure resulting in an anatomy very similar to RYGB) and control subjects, using an untargeted LC-MS/MS approach.…”