Hydrazide-based reactive matrices for the sensitive detection of aldehydes and ketones by MALDI mass spectrometry imaging.

A one-step, on-tissue chemical derivatisation method for MALDI mass spectrometry imaging was found to improve the detectability of aldehydes and ketones by charge-tagging. The developed reactive matrices, containing a UV-chromophore, ionisable moiety and hydrazide group, showed an equal or higher detection efficiency than Girard's reagent P, enabling improved imaging of brain metabolites without the need for additional co-matrices.

Regional Changes in Brain Biomolecular Markers in a Collagen-Induced Arthritis Rat Model.

BACKGROUND: The effects of collagen-induced arthritis (CIA), a model of systemic inflammation, on brain regional molecular markers associated with neurological disorders are uncertain. OBJECTIVE: This study investigated the brain regional molecular changes in markers associated with inflammation and neuronal dysfunction in a CIA model. METHODS: Fourteen male Sprague Dawley rats were divided into control (n = 5) or CIA (n = 9) groups. 10 weeks after CIA induction, brain tissue was collected. Brain regional mRNA expression of inflammatory markers (IL-1ß and IL-6), apoptotic markers (BAX and Bcl2) and neurotrophic factors (BDNF, CREB and TrkB) was determined. Monoamine distribution and abundance in different brain regions were determine by mass spectrometry imaging (MSI). RESULTS: Neuroinflammation was confirmed in the CIA group by increased IL-ß mRNA expression, concurrent with an increased BAX/Bcl2 ratio. The mRNA expression of CREB was increased in the midbrain and hippocampus while BDNF was increased and TrkB was decreased across all brain regions in CIA compared to control animals. Serotonin was decreased in the midbrain and hippocampus while dopamine was decreased in the striatum of CIA rats, compared to controls. CONCLUSION: CIA resulted in neuroinflammation concurrent with an apoptotic state and aberrant expression of neurotrophic factors and monoamines in the brain, suggestive of neurodegeneration.

Hypoxic-Ischemic Insult Alters Polyamine and Neurotransmitter Abundance in the Specific Neonatal Rat Brain Subregions.

Neonatal hypoxic-ischemic (HI) brain insult is a major cause of neonatal mortality and morbidity. To assess the underlying pathological mechanisms, we mapped the spatiotemporal changes in polyamine, amino acid, and neurotransmitter levels, following HI insult (by the Rice-Vannucci method) in the brains of seven-day-old rat pups. Matrix-assisted laser desorption/ionization mass spectrometry imaging of chemically modified small-molecule metabolites by 4-(anthracen-9-yl)-2-fluoro-1-methylpyridin-1-ium iodide revealed critical HI-related metabolomic changes of 22 metabolites in 14 rat brain subregions, much earlier than light microscopy detected signs of neuronal damage. For the first time, we demonstrated excessive polyamine oxidation and accumulation of 3-aminopropanal in HI neonatal brains, which was later accompanied by neuronal apoptosis enhanced by increases in glycine and norepinephrine in critically affected brain regions. Specifically, putrescine, cadaverine, and 3-aminopropanal increased significantly as early as 12 h postinsult, mainly in motor and somatosensory cortex, hippocampus, and midbrain, followed by an increase in norepinephrine 24 h postinsult, which was predominant in the caudate putamen, the region most vulnerable to HI. The decrease of γ-aminobutyric acid (GABA) and the continuous dysregulation of the GABAergic system together with low taurine levels up to 36 h sustained progressive neurodegenerative cellular processes. The molecular alterations presented here at the subregional rat brain level provided unprecedented insight into early metabolomic changes in HI-insulted neonatal brains, which may further aid in the identification of novel therapeutic targets for the treatment of neonatal HI encephalopathy.

Applying Spatial Metabolomics To Investigate Age- and Drug-Induced Neurochemical Changes.

In an era when population aging is increasing the burden of neurodegenerative conditions, deciphering the mechanisms underlying brain senescence is more important than ever. Here, we present a spatial metabolomics analysis of age-induced neurochemical alterations in the mouse brain using negative ionization mode mass spectrometry imaging. The age-dependent effects of the acetylcholinesterase inhibitor tacrine were simultaneously examined. For ultrahigh mass resolution analysis, we utilized a Fourier-transform ion cyclotron resonance spectrometer. To complement this, a trapped ion mobility spectrometry time-of-flight analyzer provided high speed and lateral resolution. The chosen approach facilitated the detection and identification of a wide range of metabolites, from amino acids to sphingolipids. We reported significant, age-dependent alterations in brain lipids which were most evident for sulfatides and lysophosphatidic acids. Sulfatide species, which are mainly localized to white matter, either increased or decreased with age, depending on the carbon chain length and hydroxylation stage. Lysophosphatidic acids were found to decrease with age in the detailed cortical and hippocampal subregions. An age-dependent increase in the glutamine/glutamate ratio, an indicator of glia-neuron interconnection and neurotoxicity, was detected after tacrine administration. The presented metabolic mapping approach was able to provide visualizations of the lipid signaling and neurotransmission alterations induced by early aging and can thus be beneficial to further elucidating age-related neurochemical pathways.

Hydrogen production by a fully de novo enzyme.

Molecular catalysts based on abundant elements that function in neutral water represent an essential component of sustainable hydrogen production. Artificial hydrogenases based on protein-inorganic hybrids have emerged as an intriguing class of catalysts for this purpose. We have prepared a novel artificial hydrogenase based on cobaloxime bound to a de novo three alpha-helical protein, α3C, via a pyridyl-based unnatural amino acid. The functionalized de novo protein was characterised by UV-visible, CD, and EPR spectroscopy, as well as MALDI spectrometry, which confirmed the presence and ligation of cobaloxime to the protein. The new de novo enzyme produced hydrogen under electrochemical, photochemical and reductive chemical conditions in neutral water solution. A change in hydrogen evolution capability of the de novo enzyme compared with native cobaloxime was observed, with turnover numbers around 80% of that of cobaloxime, and hydrogen evolution rates of 40% of that of cobaloxime. We discuss these findings in the context of existing literature, how our study contributes important information about the functionality of cobaloximes as hydrogen evolving catalysts in protein environments, and the feasibility of using de novo proteins for development into artificial metalloenzymes. Small de novo proteins as enzyme scaffolds have the potential to function as upscalable bioinspired catalysts thanks to their efficient atom economy, and the findings presented here show that these types of novel enzymes are a possible product.

Two Different Strategies for Stabilization of Brain Tissue and Extraction of Neuropeptides.

Neuropeptides are bioactive peptides that are synthesized and secreted by neurons in signaling pathways in the brain. Peptides and proteins are extremely vulnerable to proteolytic cleavage when their biological surrounding changes. This makes neuropeptidomics challenging due to the rapid alterations that occur to the peptidome after harvesting of brain tissue samples. For a successful neuropeptidomic study, the biological tissue sample analyzed should resemble the living state as much as possible. Heat stabilization has been proven to inhibit postmortem degradation by denaturing proteolytic enzymes, hence increasing identification rates of neuropeptides. Here, we describe two different stabilization protocols for rodent brain samples that increase the number of intact mature neuropeptides and minimize interference from degradation products of abundant proteins. Additionally, we present an extraction protocol that aims to extract a wide range of hydrophilic and hydrophobic neuropeptides by sequentially using an aqueous and an organic extraction medium.

Rapid Metabolic Profiling of 1 µL Crude Cerebrospinal Fluid by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging Can Differentiate De Novo Parkinson's Disease.

Parkinson's disease (PD) is a highly prevalent neurodegenerative disorder affecting the motor system. However, the correct diagnosis of PD and atypical parkinsonism may be difficult with high clinical uncertainty. There is an urgent need to identify reliable biomarkers using high-throughput, molecular-specific methods to improve current diagnostics. Here, we present a matrix-assisted laser desorption/ionization mass spectrometry imaging method that requires minimal sample preparation and only 1 µL of crude cerebrospinal fluid (CSF). The method enables analysis of hundreds of samples in a single experiment while simultaneously detecting numerous metabolites with subppm mass accuracy. To test the method, we analyzed CSF samples from 12 de novo PD patients (that is, newly diagnosed and previously untreated) and 12 age-matched controls. Within the identified molecules, we found neurotransmitters and their metabolites such as γ-aminobutyric acid, 3-methoxytyramine, homovanillic acid, serotonin, histamine, amino acids, and metabolic intermediates. Limits of detection were estimated for multiple neurotransmitters with high linearity (R2 > 0.99) and sensitivity (as low as 16 pg/µL). Application of multivariate classification led to a highly significant (P < 0.001) model of PD prediction with a 100% classification rate, which was further thoroughly validated with a permutation test and univariate analysis. Molecules related to the neuromelanin pathway were found to be significantly increased in the PD group, indicated by their elevated relative intensities compared to the control group. Our method enables rapid detection of PD-related biomarkers in low sample volumes and could serve as a valuable tool in the development of robust PD diagnostics.

Label-Free Quantitative Thermal Proteome Profiling Reveals Target Transcription Factors with Activities Modulated by MC3R Signaling.

Thermal proteome profiling with label-free quantitation using ion-mobility-enhanced LC-MS offers versatile data sets, providing information on protein differential expression, thermal stability, and the activities of transcription factors. We developed a multidimensional data analysis workflow for label-free quantitative thermal proteome profiling (TPP) experiments that incorporates the aspects of gene set enrichment analysis, differential protein expression analysis, and inference of transcription factor activities from LC-MS data. We applied it to study the signaling processes downstream of melanocortin 3 receptor (MC3R) activation by endogenous agonists derived from the proopiomelanocortin prohormone: ACTH, α-MSH, and γ-MSH. The obtained information was used to map signaling pathways downstream of MC3R and to deduce transcription factors responsible for cellular response to ligand treatment. Using our workflow, we identified differentially expressed proteins and investigated their thermal stability. We found in total 298 proteins with altered thermal stability, resulting from MC3R activation. Out of these, several proteins were transcription factors, indicating them as being downstream target regulators that take part in the MC3R signaling cascade. We found transcription factors CCAR2, DDX21, HMGB2, SRSF7, and TET2 to have altered thermal stability. These apparent target transcription factors within the MC3R signaling cascade play important roles in immune responses. Additionally, we inferred the activities of the transcription factors identified in our data set. This was done with Bayesian statistics using the differential expression data we obtained with label-free quantitative LC-MS. The inferred transcription factor activities were validated in our bioinformatic pipeline by the phosphorylated peptide abundances that we observed, highlighting the importance of post-translational modifications in transcription factor regulation. Our multidimensional data analysis workflow allows for a comprehensive characterization of the signaling processes downstream of MC3R activation. It provides insights into protein differential expression, thermal stability, and activities of key transcription factors. All proteomic data generated in this study are publicly available at DOI: 10.6019/PXD039945.

Spatial multimodal analysis of transcriptomes and metabolomes in tissues.

We present a spatial omics approach that combines histology, mass spectrometry imaging and spatial transcriptomics to facilitate precise measurements of mRNA transcripts and low-molecular-weight metabolites across tissue regions. The workflow is compatible with commercially available Visium glass slides. We demonstrate the potential of our method using mouse and human brain samples in the context of dopamine and Parkinson's disease.

Prosaposin maintains lipid homeostasis in dopamine neurons and counteracts experimental parkinsonism in rodents.

Prosaposin (PSAP) modulates glycosphingolipid metabolism and variants have been linked to Parkinson's disease (PD). Here, we find altered PSAP levels in the plasma, CSF and post-mortem brain of PD patients. Altered plasma and CSF PSAP levels correlate with PD-related motor impairments. Dopaminergic PSAP-deficient (cPSAPDAT) mice display hypolocomotion and depression/anxiety-like symptoms with mildly impaired dopaminergic neurotransmission, while serotonergic PSAP-deficient (cPSAPSERT) mice behave normally. Spatial lipidomics revealed an accumulation of highly unsaturated and shortened lipids and reduction of sphingolipids throughout the brains of cPSAPDAT mice. The overexpression of α-synuclein via AAV lead to more severe dopaminergic degeneration and higher p-Ser129 α-synuclein levels in cPSAPDAT mice compared to WT mice. Overexpression of PSAP via AAV and encapsulated cell biodelivery protected against 6-OHDA and α-synuclein toxicity in wild-type rodents. Thus, these findings suggest PSAP may maintain dopaminergic lipid homeostasis, which is dysregulated in PD, and counteract experimental parkinsonism.

Spatial lipidomics reveals brain region-specific changes of sulfatides in an experimental MPTP Parkinson's disease primate model.

Metabolism of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) to the neurotoxin MPP+ in the brain causes permanent Parkinson's disease-like symptoms by destroying dopaminergic neurons in the pars compacta of the substantia nigra in humans and non-human primates. However, the complete molecular pathology underlying MPTP-induced parkinsonism remains poorly understood. We used dual polarity matrix-assisted laser desorption/ionization mass spectrometry imaging to thoroughly image numerous glycerophospholipids and sphingolipids in coronal brain tissue sections of MPTP-lesioned and control non-human primate brains (Macaca mulatta). The results revealed specific distributions of several sulfatide lipid molecules based on chain-length, number of double bonds, and importantly, hydroxylation stage. More specifically, certain long-chain hydroxylated sulfatides with polyunsaturated chains in the molecular structure were depleted within motor-related brain regions in the MPTP-lesioned animals, e.g., external and internal segments of globus pallidus and substantia nigra pars reticulata. In contrast, certain long-chain non-hydroxylated sulfatides were found to be elevated within the same brain regions. These findings demonstrate region-specific dysregulation of sulfatide metabolism within the MPTP-lesioned macaque brain. The depletion of long-chain hydroxylated sulfatides in the MPTP-induced pathology indicates oxidative stress and oligodendrocyte/myelin damage within the pathologically relevant brain regions. Hence, the presented findings improve our current understanding of the molecular pathology of MPTP-induced parkinsonism within primate brains, and provide a basis for further research regarding the role of dysregulated sulfatide metabolism in PD.

On-Tissue Chemical Derivatization for Comprehensive Mapping of Brain Carboxyl and Aldehyde Metabolites by MALDI-MS Imaging.

The visualization of small metabolites by MALDI mass spectrometry imaging in brain tissue sections is challenging due to low detection sensitivity and high background interference. We present an on-tissue chemical derivatization MALDI mass spectrometry imaging approach for the comprehensive mapping of carboxyls and aldehydes in brain tissue sections. In this approach, the AMPP (1-(4-(aminomethyl)phenyl)pyridin-1-ium chloride) derivatization reagent is used for the covalent charge-tagging of molecules containing carboxylic acid (in the presence of peptide coupling reagents) and aldehydes. This includes free fatty acids and the associated metabolites, fatty aldehydes, dipeptides, neurotoxic reactive aldehydes, amino acids, neurotransmitters and associated metabolites, as well as tricarboxylic acid cycle metabolites. We performed sensitive ultrahigh mass resolution MALDI-MS detection and imaging of various carboxyl- and aldehyde-containing endogenous metabolites simultaneously in rodent brain tissue sections. We verified the AMPP-derivatized metabolites by tandem MS for structural elucidation. This approach allowed us to image numerous aldehydes and carboxyls, including certain metabolites which had been undetectable in brain tissue sections. We also demonstrated the application of on-tissue derivatization to carboxyls and aldehydes in coronal brain tissue sections of a nonhuman primate Parkinson's disease model. Our methodology provides a powerful tool for the sensitive, simultaneous spatial molecular imaging of numerous aldehydes and carboxylic acids during pathological states, including neurodegeneration, in brain tissue.

Electrochemically Etched Tapered-Tip Stainless-Steel Electrospray-Ionization Emitters for Capillary Electrophoresis-Mass Spectrometry.

We have used household consumables to facilitate electrochemical etching of stainless-steel hypodermic tubing to produce tapered-tip emitters suitable for electrospray ionization for use in mass spectrometry. The process involves the use of 1% oxalic acid and a 5 W USB power adapter, commonly known as a phone charger. Further, our method avoids the otherwise commonly used strong acids that entail chemical hazards: concentrated HNO3 for etching stainless steel, or concentrated HF for etching fused silica. Hence, we here provide a convenient and self-inhibiting procedure with minimal chemical hazards to manufacture tapered-tip stainless-steel emitters. We show its performance in metabolomic analysis with CE-MS of a tissue homogenate where the metabolites acetylcarnitine, arginine, carnitine, creatine, homocarnosine, and valerylcarnitine were identified, all with basepeak separated electropherograms, within <6 min of separation. The mass spectrometry data are freely available through the MetaboLight public data repository via access number MTBLS7230.

RNA interactome capture in Escherichia coli globally identifies RNA-binding proteins.

RNA-binding proteins (RPBs) are deeply involved in fundamental cellular processes in bacteria and are vital for their survival. Despite this, few studies have so far been dedicated to direct and global identification of bacterial RBPs. We have adapted the RNA interactome capture (RIC) technique, originally developed for eukaryotic systems, to globally identify RBPs in bacteria. RIC takes advantage of the base pairing potential of poly(A) tails to pull-down RNA-protein complexes. Overexpressing poly(A) polymerase I in Escherichia coli drastically increased transcriptome-wide RNA polyadenylation, enabling pull-down of crosslinked RNA-protein complexes using immobilized oligo(dT) as bait. With this approach, we identified 169 putative RBPs, roughly half of which are already annotated as RNA-binding. We experimentally verified the RNA-binding ability of a number of uncharacterized RBPs, including YhgF, which is exceptionally well conserved not only in bacteria, but also in archaea and eukaryotes. We identified YhgF RNA targets in vivo using CLIP-seq, verified specific binding in vitro, and reveal a putative role for YhgF in regulation of gene expression. Our findings present a simple and robust strategy for RBP identification in bacteria, provide a resource of new bacterial RBPs, and lay the foundation for further studies of the highly conserved RBP YhgF.

Enantioselective CE-MS analysis of ketamine metabolites in urine.

The chiral drug ketamine has long-lasting antidepressant effects with a fast onset and is also suitable to treat patients with therapy-resistant depression. The metabolite hydroxynorketamine (HNK) plays an important role in the antidepressant mechanism of action. Hydroxylation at the cyclohexanone ring occurs at positions 4, 5, and 6 and produces a total of 12 stereoisomers. Among those, the four 6HNK stereoisomers have the strongest antidepressant effects. Capillary electrophoresis with highly sulfated γ-cyclodextrin (CD) as a chiral selector in combination with mass spectrometry (MS) was used to develop a method for the enantioselective analysis of HNK stereoisomers with a special focus on the 6HNK stereoisomers. The partial filling approach was applied in order to avoid contamination of the MS with the chiral selector. Concentration of the chiral selector and the length of the separation zone were optimized. With 5% highly sulfated γ-CD in 20 mM ammonium formate with 10% formic acid and a 75% filling the four 6HNK stereoisomers could be separated with a resolution between 0.79 and 3.17. The method was applied to analyze fractionated equine urine collected after a ketamine infusion and to screen the fractions as well as unfractionated urine for the parent drug ketamine and other metabolites, including norketamine and dehydronorketamine.

Zero-Degree Celsius Capillary Electrophoresis Electrospray Ionization for Hydrogen Exchange Mass Spectrometry.

Currently, fast liquid chromatographic separations at low temperatures are exclusively used for the separation of peptides generated in hydrogen deuterium exchange (HDX) workflows. However, it has been suggested that capillary electrophoresis may be a better option for use with HDX. We performed in solution HDX on peptides and bovine hemoglobin (Hb) followed by quenching, pepsin digestion, and cold capillary electrophoretic separation coupled with mass spectrometry (MS) detection for benchmarking a laboratory-built HDX-MS platform. We found that capillaries with a neutral coating to eliminate electroosmotic flow and adsorptive processes provided fast separations with upper limit peak capacities surpassing 170. In contrast, uncoated capillaries achieved 30% higher deuterium retention for an angiotensin II peptide standard owing to faster separations but with only half the peak capacity of coated capillaries. Data obtained using two different separation conditions on peptic digests of Hb showed strong agreement of the relative deuterium uptake between methods. Processed data for denatured versus native Hb after deuterium labeling for the longest timepoint in this study (50,000 s) also showed agreement with subunit interaction sites determined by crystallographic methods. All proteomic data are available under DOI: 10.6019/PXD034245.

The Influence of Plant Stress Hormones and Biotic Elicitors on Cyclotide Production in Viola uliginosa Cell Suspension Cultures.

Cyclotides are macrocycle peptides produced by plants from several families, including Violaceae. These compounds have the potential for applications in medicine, bioengineering and crop protection thanks to their multiple biological activities. In most cases, cyclotides are extracted from plant material. Plant cell culture provides a viable and sustainable form of plant biomass production Cyclotides are host defense peptides. The aim of the current study was to test whether different plant stress hormones and biological elicitors have effects on cyclotide production in Viola uliginosa suspension cultures. Different concentrations of jasmonic acid (JA), salicylic acid (SA), abscisic acid (ABA) and neutralized pathogens were tested. The cyclotide production was assessed using MALDI-MS. Five major peptides produced by V. uliginosa cultures were chosen for analysis, of which one was sequenced de novo. The treatments had little influence on the suspension's growth, with the exception of 100 µM SA, which enhanced the biomass increase, and 100 µM ABA, which was toxic. Significant increases in the production of three cyclotides (viul M, cyO13 and cyO3) were observed in suspensions primed with JA (50 µM, 100 µM, 200 µM) after 14 days of culturing. Biotic elicitors had no observable effect on cyclotide production. The current study indicates that some cyclotides in V. uliginosa are triggered in response to JA. The stress plant hormones can be used to enhance plant cell culture-based production systems.

Advances in spatial mass spectrometry enable in-depth neuropharmacodynamics.

Mass spectrometry imaging (MSI) is a powerful technique that combines the ability of microscopy to provide spatial information about multiple molecular species with the specificity of mass spectrometry (MS) for unlabeled mapping of analytes in diverse biological tissues. Initial pharmacological applications focused on drug distributions in different organs, including the compartmentalized brain. However, recent technological advances in instrumentation, software, and chemical tools have allowed its use in quantitative spatial omics. It now enables visualization of distributions of diverse molecules at high lateral resolution in studies of the pharmacokinetic and neuropharmacodynamic effects of drugs on functional biomolecules. Therefore, it has become a versatile technique with a multitude of applications that have transformed neuropharmacological research and enabled research into brain physiology at unprecedented resolution, as described in this review.

Basal ganglia neuropeptides show abnormal processing associated with L-DOPA-induced dyskinesia.

L-DOPA administration is the primary treatment for Parkinson's disease (PD) but long-term administration is usually accompanied by hyperkinetic side-effects called L-DOPA-induced dyskinesia (LID). Signaling neuropeptides of the basal ganglia are affected in LID and changes in the expression of neuropeptide precursors have been described, but the final products formed from these precursors have not been well defined and regionally mapped. We therefore used mass spectrometry imaging to visualize and quantify neuropeptides in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine exposed parkinsonian and LID Macaca mulatta brain samples. We found that dyskinesia severity correlated with the levels of some abnormally processed peptides - notably, des-tyrosine dynorphins, substance P (1-7), and substance P (1-9) - in multiple brain regions. Levels of the active neuropeptides; dynorphin B, dynorphin A (1-8), α-neoendorphin, substance P (1-11), and neurokinin A, in the globus pallidus and substantia nigra correlated with putaminal levels of L-DOPA. Our results demonstrate that the abundance of selected active neuropeptides is associated with L-DOPA concentrations in the putamen, emphasizing their sensitivity to L-DOPA. Additionally, levels of truncated neuropeptides (which generally exhibit reduced or altered receptor affinity) correlate with dyskinesia severity, particularly for peptides associated with the direct pathway (i.e., dynorphins and tachykinins). The increases in tone of the tachykinin, enkephalin, and dynorphin neuropeptides in LID result in abnormal processing of neuropeptides with different biological activity and may constitute a functional compensatory mechanism for balancing the increased L-DOPA levels across the whole basal ganglia.

Well-Plate µFASP for Proteomic Analysis of Single Pancreatic Islets.

Filter-aided sample preparation (FASP) is widely used in bottom-up proteomics for tryptic digestion. However, the sample recovery yield of this method is limited by the amount of the starting material. While ∼100 ng of digested protein is sufficient for thorough protein identification, proteomic information gets lost with a protein content <10 µg due to incomplete peptide recovery from the filter. We developed and optimized a flexible well-plate µFASP device and protocol that is suitable for an ∼1 µg protein sample. In 1 µg of HeLa digest, we identified 1295 ± 10 proteins with µFASP followed by analysis with liquid chromatography-mass spectrometry. In contrast, only 524 ± 5 proteins were identified with the standard FASP protocol, while 1395 ± 4 proteins were identified in 20 µg after standard FASP as a benchmark. Furthermore, we conducted a combined peptidomic and proteomic study of single pancreatic islets with well-plate µFASP. Here, we separated neuropeptides and digested the remaining on-filter proteins for bottom-up proteomic analysis. Our results indicate inter-islet heterogeneity for the expression of proteins involved in glucose catabolism, pancreatic hormone processing, and secreted peptide hormones. We consider our method to provide a useful tool for proteomic characterization of samples where the biological material is scarce. All proteomic data are available under DOI: 10.6019/PXD029039.