An automated workflow based on data independent acquisition for practical and high-throughput personalized assay development and minimal residual disease monitoring in multiple myeloma patients.

OBJECTIVES: Minimal residual disease (MRD) status in multiple myeloma (MM) is an important prognostic biomarker. Personalized blood-based targeted mass spectrometry detecting M-proteins (MS-MRD) was shown to provide a sensitive and minimally invasive alternative to MRD-assessment in bone marrow. However, MS-MRD still comprises of manual steps that hamper upscaling of MS-MRD testing. Here, we introduce a proof-of-concept for a novel workflow using data independent acquisition-parallel accumulation and serial fragmentation (dia-PASEF) and automated data processing. METHODS: Using automated data processing of dia-PASEF measurements, we developed a workflow that identified unique targets from MM patient sera and personalized protein sequence databases. We generated patient-specific libraries linked to dia-PASEF methods and subsequently quantitated and reported M-protein concentrations in MM patient follow-up samples. Assay performance of parallel reaction monitoring (prm)-PASEF and dia-PASEF workflows were compared and we tested mixing patient intake sera for multiplexed target selection. RESULTS: No significant differences were observed in lowest detectable concentration, linearity, and slope coefficient when comparing prm-PASEF and dia-PASEF measurements of serial dilutions of patient sera. To improve assay development times, we tested multiplexing patient intake sera for target selection which resulted in the selection of identical clonotypic peptides for both simplex and multiplex dia-PASEF. Furthermore, assay development times improved up to 25× when measuring multiplexed samples for peptide selection compared to simplex. CONCLUSIONS: Dia-PASEF technology combined with automated data processing and multiplexed target selection facilitated the development of a faster MS-MRD workflow which benefits upscaling and is an important step towards the clinical implementation of MS-MRD.

The GlycoPaSER Prototype as a Real-Time N-Glycopeptide Identification Tool Based on the PaSER Parallel Computing Platform.

Real-time database searching allows for simpler and automated proteomics workflows as it eliminates technical bottlenecks in high-throughput experiments. Most importantly, it enables results-dependent acquisition (RDA), where search results can be used to guide data acquisition during acquisition. This is especially beneficial for glycoproteomics since the wide range of physicochemical properties of glycopeptides lead to a wide range of optimal acquisition parameters. We established here the GlycoPaSER prototype by extending the Parallel Search Engine in Real-time (PaSER) functionality for real-time glycopeptide identification from fragmentation spectra. Glycopeptide fragmentation spectra were decomposed into peptide and glycan moiety spectra using common N-glycan fragments. Each moiety was subsequently identified by a specialized algorithm running in real-time. GlycoPaSER can keep up with the rate of data acquisition for real-time analysis with similar performance to other glycoproteomics software and produces results that are in line with the literature reference data. The GlycoPaSER prototype presented here provides the first proof-of-concept for real-time glycopeptide identification that unlocks the future development of RDA technology to transcend data acquisition.

Quantification of 782 Plasma Peptides by Multiplexed Targeted Proteomics.

Blood analysis is one of the foundations of clinical diagnostics. In recent years, the analysis of proteins in blood samples by mass spectrometry has taken a jump forward in terms of sensitivity and the number of identified proteins. The recent development of parallel reaction monitoring with parallel accumulation and serial fragmentation (prm-PASEF) combines ion mobility as an additional separation dimension. This increases the proteome coverage while allowing the use of shorter chromatographic gradients. To demonstrate the method's full potential, we used an isotope-labeled synthetic peptide mix of 782 peptides, derived from 579 plasma proteins, spiked into blood plasma samples with a prm-PASEF measurement allowing the quantification of 565 plasma proteins by targeted proteomics. As a less time-consuming alternative to the prm-PASEF method, we describe guided data independent acquisition (dia)-PASEF (g-dia-PASEF) and compare its application to prm-PASEF for measuring blood plasma. To demonstrate both methods' performance in clinical samples, 20 patient plasma samples from a colorectal cancer (CRC) cohort were analyzed. The analysis identified 14 differentially regulated proteins between the CRC patient and control individual plasma samples. This shows the technique's potential for the rapid and unbiased screening of blood proteins, abolishing the need for the preselection of potential biomarker proteins.

Novel canonical and non-canonical viral antigens extend current targets for immunotherapy of HPV-driven cervical cancer.

Current immunotherapeutic approaches for human papillomavirus (HPV)-driven cervical cancer target the viral oncogenes E6 and E7. We report viral canonical and alternative reading frame (ARF)-derived sequences presented on cervical tumor cells, including antigens encoded by the conserved viral gene E1. We confirm immunogenicity of the identified viral peptides in HPV-positive women, and women with cervical intraepithelial neoplasia. We observe consistent transcription of the E1, E6, and E7 genes in 10 primary cervical tumor resections from the four most common high-risk HPV subtypes (HPV16, 18, 31, and 45), suggesting the suitability of E1 as therapeutic target. We finally confirm HLA presentation of canonical peptides derived from E6 and E7, and ARF-derived viral peptides from a reverse-strand transcript spanning the HPV E1 and E2 genes in primary human cervical tumor tissue. Our results extend currently known viral immunotherapeutic targets in cervical cancer and highlight E1 as an important cervical cancer antigen.

Oxonium Ion-Guided Optimization of Ion Mobility-Assisted Glycoproteomics on the timsTOF Pro.

Spatial separation of ions in the gas phase, providing information about their size as collisional cross-sections, can readily be achieved through ion mobility. The timsTOF Pro (Bruker Daltonics) series combines a trapped ion mobility device with a quadrupole, collision cell, and a time-of-flight analyzer to enable the analysis of ions at great speed. Here, we show that the timsTOF Pro is capable of physically separating N-glycopeptides from nonmodified peptides and producing high-quality fragmentation spectra, both beneficial for glycoproteomics analyses of complex samples. The glycan moieties enlarge the size of glycopeptides compared with nonmodified peptides, yielding a clear cluster in the mobilogram that, next to increased dynamic range from the physical separation of glycopeptides and nonmodified peptides, can be used to make an effective selection filter for directing the mass spectrometer to analytes of interest. We designed an approach where we (1) focused on a region of interest in the ion mobilogram and (2) applied stepped collision energies to obtain informative glycopeptide tandem mass spectra on the timsTOF Pro:glyco-polygon-stepped collision energy-parallel accumulation serial fragmentation. This method was applied to selected glycoproteins, human plasma- and neutrophil-derived glycopeptides. We show that the achieved physical separation in the region of interest allows for improved extraction of information from the samples, even at shorter liquid chromatography gradients of 15 min. We validated our approach on human neutrophil and plasma samples of known makeup, in which we captured the anticipated glycan heterogeneity (paucimannose, phosphomannose, high mannose, hybrid and complex glycans) from plasma and neutrophil samples at the expected abundances. As the method is compatible with off-the-shelve data acquisition routines and data analysis software, it can readily be applied by any laboratory with a timsTOF Pro and is reproducible as demonstrated by a comparison between two laboratories.

HLAII peptide presentation of infliximab increases when complexed with TNF.

CD4+ T-cell activation through recognition of Human Leukocyte Antigen II (HLAII)-presented peptides is a key step in the development of unwanted immune response against biotherapeutics, such as the generation of anti-drug antibodies (ADA). Therefore, the identification of HLAII-presented peptides derived from biotherapeutics is a crucial part of immunogenicity risk assessment and mitigation strategies during drug development. To date, numerous CD4+ T-cell epitopes have been identified by HLAII immunopeptidomics in antibody-based biotherapeutics using either their native or aggregated form. Antibody-target immune complexes have been detected in patients with ADA and are thought to play a role in ADA development by enhancing the presentation of CD4+ T-cell epitopes at the surface of antigen presenting cells (APCs). The aim of this study was to investigate the effect of biotherapeutic antibody-target immune complexes on the HLAII peptide presentation of biotherapeutics in human primary monocyte-derived dendritic cells (DCs). The trimeric tumor necrosis factor (TNF) and its biotherapeutic antagonists infliximab (INFL), adalimumab (ADAL), and a single armed Fab' were used as a model system. The HLAII immunopeptidome of DCs loaded with antagonists or their immune complexes with TNF was analyzed by trapped ion mobility time-of-flight mass spectrometry (timsTOF MS) leading to the identification of ~ 12,000 unique HLAII-associated peptides per preparation. Anti-TNF sequences were detected at a median of 0.3% of the total immunopeptidome, against a majority background of peptides from endogenous and media-derived proteins. TNF antagonist presentation spanned the variable and constant regions in a widespread manner in both light and heavy chains, consistent with previously discovered HLAII peptides. This investigation extends the collection of observed HLAII peptides from anti-TNF biotherapeutics to include sequences that at least partially span the complementary determining regions (CDRs), such as the LCDR1 for both INFL and ADAL. Although antagonist presentation varied significantly across donors, peptides from both bivalent antagonists INFL and ADAL were more highly presented relative to the Fab'. While TNF immune complexes did not alter overall HLAII presentation, a moderate increase in presentation of a subset of peptide clusters was observed in the case of INFL-TNF, which included HCDR2, HCDR3 and LCDR2 sequences.

On-Chip Preconcentration Microchip Capillary Electrophoresis Based CE-PRM-LIVE for High-Throughput Selectivity Profiling of Deubiquitinase Inhibitors.

The family of deubiquitinases (DUBs) comprises ∼100 enzymes that cleave ubiquitin from substrate proteins and thereby regulate key aspects of human physiology. DUBs have recently emerged as disease-relevant and chemically tractable, although currently there are no approved DUB-targeting drugs and most preclinical small molecules are low-potency and/or multitargeted. We paired a novel capillary electrophoresis microchip containing an integrated, "on-chip" C18 bed (SPE-ZipChip) with a TMT version of our recently described PRM-LIVE acquisition scheme on a timsTOF Pro mass spectrometer to facilitate rapid activity-based protein profiling of DUB inhibitors. We demonstrate the ability of the SPE-ZipChip to improve proteome coverage of complex samples as well as the quantitation integrity of CE-PRM-LIVE for TMT labeled samples. These technologies provide a platform to accurately quantify competitive binding of covalent and reversible inhibitors in a multiplexed assay that spans 49 endogenous DUBs in less than 15 min.

The Parallel Reaction Monitoring-Parallel Accumulation-Serial Fragmentation (prm-PASEF) Approach for Multiplexed Absolute Quantitation of Proteins in Human Plasma.

Mass spectrometry (MS)-based quantitative proteomic methods have become some of the major tools for protein biomarker discovery and validation. The recently developed parallel reaction monitoring-parallel accumulation-serial fragmentation (prm-PASEF) approach on a Bruker timsTOF Pro mass spectrometer allows the addition of ion mobility as a new dimension to LC-MS-based proteomics and increases proteome coverage at a reduced analysis time. In this study, a prm-PASEF approach was used for the multiplexed absolute quantitation of proteins in human plasma using isotope-labeled peptide standards for 125 plasma proteins, over a broad (104-106) dynamic range. Optimization of LC and MS parameters, such as accumulation time and collision energy, resulted in improved sensitivity for more than half of the targets (73 out of 125 peptides) by increasing the signal-to-noise ratio by a factor of up to 10. Overall, 41 peptides showed up to a 2-fold increase in sensitivity, 25 peptides showed up to a 5-fold increase in sensitivity, and 7 peptides showed up to a 10-fold increase in sensitivity. Implementation of the prm-PASEF method allowed absolute protein quantitation (down to 1.13 fmol) in human plasma samples. A comparison of the concentration values of plasma proteins determined by MRM on a QTRAP instrument and by prm-PASEF on a timsTOF Pro revealed an excellent correlation (R2 = 0.97) with a slope of close to 1 (0.99), demonstrating that prm-PASEF is well suited for "absolute" quantitative proteomics.

Hydroxyl radical footprinting analysis of a human haptoglobin-hemoglobin complex.

Methods of structural mass spectrometry have become more popular to study protein structure and dynamics. Among them, fast photochemical oxidation of proteins (FPOP) has several advantages such as irreversibility of modifications and more facile determination of the site of modification with single residue resolution. In the present study, FPOP analysis was applied to study the hemoglobin (Hb) - haptoglobin (Hp) complex allowing identification of respective regions altered upon the complex formation. FPOP footprinting using a timsTOF Pro mass spectrometer revealed structural information for 84 and 76 residues in Hp and Hb, respectively, including statistically significant differences in the modification extent below 0.3%. The most affected residues upon complex formation were Met76 and Tyr140 in Hbα, and Tyr280 and Trp284 in Hpß. The data allowed determination of amino acids directly involved in Hb - Hp interactions and those located outside of the interaction interface yet affected by the complex formation. Also, previously modeled interaction between Hb ßTrp37 and Hp ßPhe292 was not confirmed by our data. Data are available via ProteomeXchange with identifier PXD021621.

PRM-LIVE with Trapped Ion Mobility Spectrometry and Its Application in Selectivity Profiling of Kinase Inhibitors.

Parallel reaction monitoring (PRM) has emerged as a popular approach for targeted protein quantification. With high ion utilization efficiency and first-in-class acquisition speed, the timsTOF Pro provides a powerful platform for PRM analysis. However, sporadic chromatographic drift in peptide retention time represents a fundamental limitation for the reproducible multiplexing of targets across PRM acquisitions. Here, we present PRM-LIVE, an extensible, Python-based acquisition engine for the timsTOF Pro, which dynamically adjusts detection windows for reproducible target scheduling. In this initial implementation, we used iRT peptides as retention time standards and demonstrated reproducible detection and quantification of 1857 tryptic peptides from the cell lysate in a 60 min PRM-LIVE acquisition. As an application in functional proteomics, we use PRM-LIVE in an activity-based protein profiling platform to assess binding selectivity of small-molecule inhibitors against 220 endogenous human kinases.

Correction to "Benefits of Ion Mobility Separation and Parallel Accumulation-Serial Fragmentation Technology on timsTOF Pro for the Needs of Fast Photochemical Oxidation of Protein Analysis".

[This corrects the article DOI: 10.1021/acsomega.1c00732.].

Benefits of Ion Mobility Separation and Parallel Accumulation-Serial Fragmentation Technology on timsTOF Pro for the Needs of Fast Photochemical Oxidation of Protein Analysis.

Fast photochemical oxidation of proteins (FPOP) is a recently developed technique for studying protein folding, conformations, interactions, etc. In this method, hydroxyl radicals, usually generated by KrF laser photolysis of H2O2, are used for irreversible labeling of solvent-exposed side chains of amino acids. Mapping of the oxidized residues to the protein's structure requires pinpointing of modifications using a bottom-up proteomic approach. In this work, a quadrupole time-of-flight (QTOF) mass spectrometer coupled with trapped ion mobility spectrometry (timsTOF Pro) was used for identification of oxidative modifications in a model protein. Multiple modifications on the same residues, including six modifications of histidine, were successfully resolved. Moreover, parallel accumulation-serial fragmentation (PASEF) technology allows successful sequencing of even minor populations of modified peptides. The data obtained indicate a clear improvement of the quality of the FPOP analysis from the viewpoint of the number of identified peptides bearing oxidative modifications and their precise localization. Data are available via ProteomeXchange with identifier PXD020509.

Towards quality assurance and quality control in untargeted metabolomics studies.

We describe here the agreed upon first development steps and priority objectives of a community engagement effort to address current challenges in quality assurance (QA) and quality control (QC) in untargeted metabolomic studies. This has included (1) a QA and QC questionnaire responded to by the metabolomics community in 2015 which recommended education of the metabolomics community, development of appropriate standard reference materials and providing incentives for laboratories to apply QA and QC; (2) a 2-day 'Think Tank on Quality Assurance and Quality Control for Untargeted Metabolomic Studies' held at the National Cancer Institute's Shady Grove Campus and (3) establishment of the Metabolomics Quality Assurance and Quality Control Consortium (mQACC) to drive forward developments in a coordinated manner.

Identification of multiple proteoforms biomarkers on clinical samples by routine Top-Down approaches.

Top-Down approaches have an extremely high biological relevance, especially when it comes to biomarker discovery, but the necessary pre-fractionation constraints are not easily compatible with the robustness requirements and the size of clinical sample cohorts. We have demonstrated that intact protein profiling studies could be run on UHR-Q-ToF with limited pre-fractionation (Schmit et al., 2017) [1]. The dataset presented herein is an extension of this research. Proteoforms known to play a role in the pathophysiology process of Alzheimer's disease were identified as candidate biomarkers. In this article, mass spectrometry performance of these candidates are demonstrated.

Towards a routine application of Top-Down approaches for label-free discovery workflows.

Thanks to proteomics investigations, our vision of the role of different protein isoforms in the pathophysiology of diseases has largely evolved. The idea that protein biomarkers like tau, amyloid peptides, ApoE, cystatin, or neurogranin are represented in body fluids as single species is obviously over-simplified, as most proteins are present in different isoforms and subjected to numerous processing and post-translational modifications. Measuring the intact mass of proteins by MS has the advantage to provide information on the presence and relative amount of the different proteoforms. Such Top-Down approaches typically require a high degree of sample pre-fractionation to allow the MS system to deliver optimal performance in terms of dynamic range, mass accuracy and resolution. In clinical studies, however, the requirements for pre-analytical robustness and sample size large enough for statistical power restrict the routine use of a high degree of sample pre-fractionation. In this study, we have investigated the capacities of current-generation Ultra-High Resolution Q-Tof systems to deal with high complexity intact protein samples and have evaluated the approach on a cohort of patients suffering from neurodegenerative disease. Statistical analysis has shown that several proteoforms can be used to distinguish Alzheimer disease patients from patients suffering from other neurodegenerative disease. SIGNIFICANCE: Top-down approaches have an extremely high biological relevance, especially when it comes to biomarker discovery, but the necessary pre-fractionation constraints are not easily compatible with the robustness requirements and the size of clinical sample cohorts. We have demonstrated that intact protein profiling studies could be run on UHR-Q-ToF with limited pre-fractionation. The proteoforms that have been identified as candidate biomarkers in the-proof-of concept study are derived from proteins known to play a role in the pathophysiology process of Alzheimer disease.

Quantification of a proteotypic peptide from protein C inhibitor by liquid chromatography-free SISCAPA-MALDI mass spectrometry: application to identification of recurrence of prostate cancer.

BACKGROUND: Biomarker validation remains one of the most challenging constraints to the development of new diagnostic assays. To facilitate biomarker validation, we previously developed a chromatography-free stable isotope standards and capture by antipeptide antibodies (SISCAPA)-MALDI assay allowing rapid, high-throughput quantification of protein analytes in large sample sets. Here we applied this assay to the measurement of a surrogate proteotypic peptide from protein C inhibitor (PCI) in sera from patients with prostate cancer. METHODS: A 2-plex SISCAPA-MALDI assay for quantification of proteotypic peptides from PCI and soluble transferrin receptor (sTfR) was used to measure these peptides in 159 trypsin-digested sera collected from 51 patients with prostate cancer. These patients had been treated with radiation with or without neoadjuvant androgen deprivation. RESULTS: Patients who experienced biochemical recurrence of prostate cancer showed decreased serum concentrations of the PCI peptide analyte within 18 months of treatment. The PCI peptide concentrations remained increased in the sera of patients who did not experience cancer recurrence. Prostate-specific antigen concentrations had no predictive value during the same time period. CONCLUSIONS: The high-throughput, liquid chromatography-free SISCAPA-MALDI assay is capable of rapid quantification of proteotypic PCI and sTfR peptide analytes in complex serum samples. Decreased serum concentrations of the PCI peptide were found to be related to recurrence of prostate cancer in patients treated with radiation with or without hormone therapy. However, a larger cohort of patients will be required for unequivocal validation of the PCI peptide as a biomarker for clinical use.

Precision of heavy-light peptide ratios measured by maldi-tof mass spectrometry.

We have investigated the precision of peptide quantitation by MALDI-TOF mass spectrometry (MS) using six pairs of proteotypic peptides (light) and same-sequence stable isotope labeled synthetic internal standards (heavy). These were combined in two types of dilution curves spanning 100-fold and 2000-fold ratios. Coefficients of variation (CV; standard deviation divided by mean value) were examined across replicate MALDI spots using a reflector acquisition method requiring 100 000 counts for the most intense peak in each summed spectrum. The CV of light/heavy peptide centroid peak area ratios determined on four replicate spots per sample, averaged across 11 points of a 100-fold dilution curve and over all six peptides, was 2.2% (ranging from 1.5 to 3.7% among peptides) at 55 fmol total (light + heavy) of each peptide applied per spot, and 2.5% at 11 fmol applied. The average CV of measurements at near-equivalence (light = heavy, the center of the dilution curve) for the six peptides was 1.0%, about 17-fold lower CV than that observed when five peptides were ratioed to a sixth peptide (i.e., a different-sequence internal standard). Response curves across the 100-fold range were not completely linear but could be closely modeled by a power law fit giving R(2) values >0.998 for all peptides. The MALDI-TOF MS method was used to determine the endogenous level of a proteotypic peptide (EDQYHYLLDR) of human protein C inhibitor (PCI) in a plasma digest after enrichment by capture on a high affinity antipeptide antibody, a technique called stable isotope standards and capture by anti-peptide antibodies (SISCAPA). The level of PCI was determined to be 770 ng/mL with a replicate measurement CV of 1.5% and a >14 000-fold target enrichment via SISCAPA-MALDI-TOF. These results indicate that MALDI-TOF technology can provide precise quantitation of high-to-medium abundance peptide biomarkers over a 100-fold dynamic range when ratioed to same-sequence labeled internal standards and enriched to near purity by specific antibody capture. The robustness and throughput of MALDI-TOF in comparison to conventional nano-LC-MS technology could enable currently impractical large-scale verification studies of protein biomarkers.

Automated ambient desorption-ionization platform for surface imaging integrated with a commercial Fourier transform ion cyclotron resonance mass spectrometer.

A fully automated atmospheric pressure ionization platform has been built and coupled with a commercial high-resolution Fourier transform ion cyclotron resonance mass spectrometer (FTICR-MS) instrument. The outstanding performance of this instrument allowed screening on the basis of exact masses in imaging mode. The main novel aspect was in the integration of the atmospheric pressure ionization imaging into the current software for matrix-assisted laser desorption ionization (MALDI) imaging, which allows the user of this commercial dual-source mass spectrometer to perform MALDI-MS and different ambient MS imaging from the same user interface and to utilize the same software tools. Desorption electrospray ionization (DESI) and desorption atmospheric pressure photoionization (DAPPI) were chosen to test the ambient surface imaging capabilities of this new ionization platform. Results of DESI imaging experiments performed on brain tissue sections are in agreement with previous MS imaging reports obtained by DESI imaging, but due to the high resolution and mass accuracy of the FTICR instrument it was possible to resolve several ions at the same nominal mass in the DESI-MS spectra of brain tissue. These isobaric interferences at low resolution are due to the overlap of ions from different lipid classes with different biological relevance. It was demonstrated that with the use of high-resolution MS fast imaging screening of lipids can be achieved without any preseparation steps. DAPPI, which is a relatively new and less developed ambient ionization technique compared to DESI, was used in imaging mode for the first time ever. It showed promise in imaging of phytocompounds from plant leaves, and selective ionization of a sterol lipid was achieved by DAPPI from a brain tissue sample.

Capillary electrophoresis-fourier transform ion cyclotron resonance mass spectrometry for the identification of cationic metabolites via a pH-mediated stacking-transient isotachophoretic method.

Capillary electrophoresis-mass spectrometry (CE-MS) is still widely regarded as an emerging tool in the field of metabolomics and metabolite profiling. A major reason for this is a reported lack of sensitivity of CE-MS when compared to gas chromatography-mass spectrometry GC/MS and liquid chromatography-mass spectrometry. The problems caused by the lack of sensitivity are exacerbated when CE is coupled to Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS), due to the relatively low data acquisition rate of FT-ICR MS. Here, we demonstrate the use of an online CE sample preconcentration method that uses a combination of pH-mediated stacking and transient isotachophoresis, coupled with FT-ICR MS to improve the overall detection of cationic metabolites in the bacterium Desulfovibrio vulgaris Hildenborough. This method showed a significant increase in signal-to-noise ratio when compared to CE normal sample stacking, while providing good separation efficiency, reproducibility, and linearity. Detection limits for selected amino acids were between 0.1 and 2 microM. Furthermore, FT-ICR MS detection consistently demonstrated good mass resolution and sub-ppm mass accuracy.

Characterization of DESI-FTICR mass spectrometry - from ECD to accurate mass tissue analysis.

Implementation of desorption electrospray ionization (DESI) technique on a 9.4 T Fourier transform ion cyclotron resonance (FTICR) mass spectrometer is described. Desorption electrospray technique is capable of the direct investigation of natural samples without any need for sample preparation or chromatographic separation. Since the DESI mass spectra of natural samples are very complex owing to the lack of preseparation or cleanup, the ideal mass spectrometric analyzer for these applications is a high-resolution instrument such as FTICR mass spectrometer. DESI was implemented by constructing an electronically controlled source framework comprising six linear moving stages and one rotating stage. A three-dimensional linear stage was used to accommodate samples, while another 3D linear stage equipped with rotating stage was used as a spray mount. A modified electrosonic sprayer was used as a primary electrospray device. DESI-FTICR setup was characterized with regard to geometrical, electrical and flow conditions using deposited peptide samples in range of 1-100 pmol gross deposited amount on glass and polymer surfaces. Optimized conditions enabled the routine acquisition of DESI-MS spectra on the instrument at 130 000 resolution in the broadband mode and with comparable sensitivity to data reported in the literature. Since the main significance of DESI-FTICR MS is the combination of intact tissue analysis, the capabilities of the technique were demonstrated by analyzing murine liver samples. Presence of lysophospholipids in the liver tissue was tentatively associated with the lipid metabolism taking place in liver. DESI-FTICR is also a promising technique in the field of peptide analysis due to capability of top-down sequencing using electron capture dissociation. As a proof-of-principle experiment, a small synthetic polypeptide containing 36 amino acids was ionized using DESI and was sequenced in the FTICR by means of ECD (electron capture dissociation) fragmentation. Spectra gave almost full sequence information in agreement with the known amino acid sequence of the species.