Peptidomic analysis of endogenous plasma peptides from patients with pancreatic neuroendocrine tumours

Rationale

Diagnosis of pancreatic neuroendocrine tumours requires the study of patient plasma with multiple immunoassays, using multiple aliquots of plasma. The application of mass spectrometry based techniques could reduce the cost and amount of plasma required for diagnosis.

Methods

Plasma samples from two patients with pancreatic neuroendocrine tumours were extracted using an established acetonitrile‐based plasma peptide enrichment strategy. The circulating peptidome was characterised using nano and high flow rate liquid chromatography/mass spectrometry (LC/MS) analyses. To assess the diagnostic potential of the analytical approach, a large sample batch (68 plasmas) from control subjects, and aliquots from subjects harbouring two different types of pancreatic neuroendocrine tumour (insulinoma and glucagonoma), were analysed using a 10‐min LC/MS peptide screen.

Results

The untargeted plasma peptidomics approach identified peptides derived from the glucagon prohormone, chromogranin A, chromogranin B and other peptide hormones and proteins related to control of peptide secretion. The glucagon prohormone derived peptides that were detected were compared against putative peptides that were identified using multiple antibody pairs against glucagon peptides. Comparison of the plasma samples for relative levels of selected peptides showed clear separation between the glucagonoma and the insulinoma and control samples.

Conclusions

The combination of the organic solvent extraction methodology with high flow rate analysis could potentially be used to aid diagnosis and monitor treatment of patients with functioning pancreatic neuroendocrine tumours. However, significant validation will be required before this approach can be clinically applied.

     

Study of the human plasma proteome of rheumatoid arthritis

In this study, we report a combined proteomic and peptidomic analysis of human plasma from patients with rheumatoid arthritis (RA) and controls. We used molecular weight cut-off filters (MWCO: 10 kDa) to enrich low-molecular-weight (LMW) peptides from human plasma. The peptide fraction was analyzed without trypsin digestion by capillary reversed-phase high-performance liquid chromatography (HPLC) coupled to a linear ion trap–FT-MS system, which identified 771 unique peptides in the peptidome study (from 145 protein progenitors). An anti-albumin/anti-IgG column was used to remove albumin and immunoglobulin G (IgG) from the human plasma. After that, the albumin/IgG-depleted sample was fractionated into a bound fraction and an unbound fraction on a multi-lectin affinity column (M-LAC). LC–MS analysis of the corresponding tryptic digests identified 308 proteins using the M-LAC approach. Relative differences in the following protein classifications were observed in the RA human plasma samples compared with controls: structural proteins, immuno-related proteins, protease inhibitors, coagulation proteins, transport proteins and apolipoproteins. While some of these proteins/peptides have been previously reported to be associated with RA disease such as calgranulin A, B, C and C-reactive protein, several others were newly identified (such as thymosin β4, actin, tubulin, and vimentin), which may further the understanding of the disease pathogenesis. Moreover, we have found that the peptidomic and glycoproteomic approaches were complementary and allow a more complete picture of the human plasma proteome which can be valuable in studies of disease etiology.     

Peptidomic analysis of the larval Drosophila melanogaster central nervous system by two-dimensional capillary liquid chromatography quadrupole time-of-flight mass spectrometry

Peptides are the largest class of signalling molecules found in animals. Nevertheless, in most proteomic studies peptides are overlooked since they literally fall through the mazes of the net. In analogy with proteomics technology, where all proteins expressed in a cell or tissue are analyzed, the peptidomic approach aims at the simultaneous visualization and identification of the whole peptidome of a cell or tissue, i.e. all expressed peptides with their post-translational modifications. In this paper we describe the analysis of the larval fruit fly central nervous system using two-dimensional capillary liquid chromatography/quadrupole time-of-flight tandem mass spectrometry (LC/Q-TOF-MS/MS. Using the central nervous systems of only 50 larval Drosophila as starting material, we identified 38 peptides in a single analysis, 20 of which were not detected in a previous study that reported on the one-dimensional capillary LC/MS/MS analysis of the same tissue. Among the 38 sequenced peptides, some originate from precursors, such as the tachykinin and the IFamide precursor that were entirely missed in the first study. This clearly demonstrates that the two-dimensional capillary LC approach enhances the coverage of the peptidomic analysis.     

Enrichment of peptides in serum by C(8)-functionalized magnetic nanoparticles for direct matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis

Human serum contains a complex array of proteolytically derived peptides (serum peptidome), which contain biomarkers of preclinical screening and disease diagnosis. Recently, commercial C(8)-functionalized magnetic beads (1-10 microm) were widely applied to the separation and enrichment of peptides in human serum, prior to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis. In this work, laboratory-prepared C(8)-functionalized magnetic nanoparticles (about 50 nm) were prepared and applied to the fast separation and the enrichment of peptides from serum. At first, the C(8)-magnetic nanoparticles were synthesized by modifying amine-functionalized magnetic nanoparticles with chlorodimethyloctylsilane. These synthesized C(8)-amine-functionalized magnetic particles have excellent magnetic responsibility, high dispersibility and large surface area. Finally, the C(8)-magnetic nanoparticles were successfully applied to fast and efficient enrichment of low-abundance peptides from protein tryptic digestion and human serum followed by MALDI-TOF-MS analysis.     

Prerequisites for peptidomic analysis of blood samples: I. Evaluation of blood specimen qualities and determination of technical performance characteristics

Proteomics studies aiming at a detailed analysis of proteins, and peptidomics, aiming at the analysis of the low molecular weight proteome (peptidome) offer a promising approach to discover novel biomarkers valuable for different crucial steps in detection, prevention and treatment of disease. Much emphasis has been given to the analysis of blood, since this source would by far offer the largest number of meaningful biomarker applications. Blood is a complex liquid tissue that comprises cells and extra-cellular fluid. The choice of suitable specimen collection is crucial to minimize artificial occurring processes during specimen collection and preparation (e.g. cell lysis, proteolysis). After specimen collection, sample preparation for peptidomics is carried out by physical methods (filtration, gel-chromatography, precipitation) which allow for separation based on molecular size, with and without immunodepletion of major abundant proteins. Differential Peptide Display (DPD) is an offline-coupled combination of Reversed-Phase-HPLC and MALDI mass spectrometry in combination with in-house developed data display and analysis tools. Identifications of peptides are carried out by additional mass spectrometric methods (e.g. online LC-ESI-MS/MS). In the work presented here, insights into semi-quantitative mass spectrometric profiling of plasma peptides by DPD are given. This includes proper specimen selection (plasma vs. serum), sample preparation, especially peptide extraction, the determination of sensitivity (i.e. by establishing detection limits of exogenously spiked peptides), the reproducibility for individual as well as for all peptides (Coefficient of Variation calculations) and quantification (correlation between signal intensity and concentration). Finally, the implications for clinical peptidomics are discussed.     

On-line turbulent-flow chromatography-high-performance liquid chromatography-mass spectrometry for fast sample preparation and quantitation

A sensitive and selective liquid chromatography-mass spectrometry method has been developed for the simultaneous identification and quantitation of drug substances and metabolites in rat plasma. The method combines on-line turbulent-flow chromatography, high-performance liquid chromatography and mass spectrometry. This combination is considered to be a new approach suitable for fast bio-analysis in drug discovery. Dextromethorphan, and its two metabolites, dextrorphan and 3-methoxymorphinan served as model substances. The analytes present in plasma were collected on a Cyclone column using turbulent-flow chromatography and were subsequently transferred on-line to and focused on an X-Terra MS C8 column. The analytes were eluted by a linear gradient and detected by a fast scanning mass spectrometer. The detector response was quadratic and the dynamic range was estimated to be 0.5-100 ng/ml plasma or 12.5 pg to 2.50 ng injected into the system.     

Mass spectrometric analysis of the individual variability of Bothrops jararaca venom peptide fraction. Evidence for sex-based variation among the bradykinin-potentiating peptides

Variation in the snake venom proteome is well documented and it is a ubiquitous phenomenon at all taxonomical levels. However, variation in the snake venom peptidome is so far not described. In this work we used mass spectrometry [liquid chromatography/mass spectrometry (LC/MS) and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOFMS)] to explore sex-based differences among the venom peptides of eighteen sibling specimens of Bothrops jararaca of a single litter born and raised in the laboratory. MALDI-TOFMS analyses showed individual variability among the bradykinin-potentiating peptides (BPPs), and, interestingly, four new peptides were detected only in female venoms and identified by de novo sequencing as cleaved BPPs lacking the C-terminal Q-I-P-P sequence. Similar results were obtained with venom from wild-caught adult non-sibling specimens of B. jararaca and in this case we were able to identify the gender of the specimen by analyzing the MALDI-TOF profile of the peptide fraction and finding the cleaved peptides only in female venoms. Synthetic replicates of the cleaved BPPs were less potent than the full-length BPP-10c in potentiating the bradykinin hypotensive effect, suggesting that the C-terminus is critical for the interaction of the BPPs with their mammalian molecular targets. This work represents a comprehensive mass spectrometric analysis of the peptide fraction of B. jararaca venom and shows for the first time sex-based differences in the snake venom peptidome of sibling and non-sibling snakes and suggests that the BPPs may follow distinct processing pathways in female and male individuals.     

Liquid chromatography-mass spectrometry in metabolic research. I. Metabolites of benzbromarone in human plasma and urine.

Seven benzbromarone metabolites were identified in human plasma and urine by electron-impact mass spectrometry after semipreparative high-performance liquid chromatographic fractionation and/or by liquid chromatography-mass spectrometry using a thermospray interface. The major metabolite in plasma and urine was a hydroxybenzofuranoyl species; the 1-hydroxyethyl entity was identified as a minor metabolite. Five urinary metabolites occurred in trace amounts, all of them carrying OH and/or C = O groups in different positions. The hydroxybenzofuranoyl metabolite has often been mistaken for benzarone in previous studies.     

Selective on-line serum peptide extraction and multidimensional separation by coupling a restricted-access material-based capillary trap column with nanoliquid chromatography–tandem mass spectrometry

As the serum peptidome gets increasing attention for biomarker discovery, one of the important issues is how to efficiently extract the peptides from highly complex human serum for peptidome analysis. Here we developed a fully automated platform for direct injection, on-line extraction, multidimensional separation and MS detection of peptides present in human serum. A capillary SPE column packed with a novel mix mode restricted access material (RAM) exhibiting strong cation exchange and size exclusion chromatography (SCX/SEC) properties were coupled with a nanoliquid chromatography–mass spectrometry (nanoLC-MS) system. The capillary SPE column excludes the high abundant serum proteins such as HSA by size exclusion chromatography and simultaneously extracts the low molecular weight peptides by binding to sulfonic acid residues. Subsequently, the trapped peptides are eluted to a capillary LC column packed with a RP-C18 stationary phase. After injection of only 2 μL human serum to the one-dimensional nanoLC-MS system around 400 peptides could be identified. When conducting a multidimensional separation, the described SCX/SEC/RP-MS platform allows the separation and identification of 1286 peptides present in human serum by the injection and on-line processing of 20 μL human serum sample.     

Preparation of phenyl‐functionalized magnetic mesoporous silica microspheres for the fast separation and selective enrichment of phenyl‐containing peptides

Peptide enrichment before mass spectrometry analysis is essential for large‐scale peptidomic studies, but challenges still remain. Herein, magnetic mesoporous silica microspheres with phenyl group modified interior pore walls were prepared by a facile sol–gel coating strategy, and were successfully applied for selective enrichment of phenyl‐containing peptides in complex biological samples. The newly prepared nanomaterials possessed abundant silanol groups in the exterior surface and numerous phenyl groups in the interior pore walls, as well as a large surface area (592.6 m²/g), large pore volume (0.33 cm³/g), uniform mesopores (3.8 nm), strong magnetic response (29.3 emu/g), and good dispersibility in aqueous solution. As a result of the unique structural properties and size‐exclusion effect, the core–shell phenyl‐functionalized magnetic mesoporous silica microspheres exhibited excellent performance in fast separation and selective enrichment of phenyl‐containing peptides, and the adsorption capacity for bradykinin reached 22.55 mg/g. In addition, selective enrichment of phenyl‐containing peptides from complex samples that are consist of peptides, large proteins, and human serum were achieved by using the as‐prepared microspheres, followed by high‐performance liquid chromatography with ultraviolet detection and electrospray ionization quadrupole time‐of‐flight mass spectrometry analysis. These results demonstrated the as‐prepared microspheres would be a potential candidate for endogenous phenyl‐containing peptides enrichment and biomarkers discovery in peptidome analysis.     

Sequence analysis of derivatized peptides by high-performance liquid chromatography-mass spectrometry

N-Acetyl-N,O,S-permethylated derivatives of oligopeptides were analyzed by high-performance liquid chromatography-mass spectrometry (HPLC-MS) using a moving belt interface. A heated-gas nebulizer was employed for sample deposition, thus permitting the effective use of a water-methanol gradient covering the range from 5% to 95% water at mobile phase flow-rates of 0.5 ml/min. We demonstrate in this paper that it is possible to sequence the octapeptide derived from the C-chain of glucagon by HPLC-MS analysis of a permethylated enzymatic hydrolysate of this peptide using overlap information from the mass spectral patterns. Moreover, it is shown that peptides not readily amenable to analysis by gas chromatography-MS can be analyzed using this approach. Preliminary results suggest that N-acetyl-methyl ester derivatives of oligopeptides may in specific cases also be a useful alternative for HPLC-MS analysis of complex oligopeptide mixtures.     

Analysis of the low molecular weight serum peptidome using ultrafiltration and a hybrid ion trap-Fourier transform mass spectrometer

Advances in proteomics are continuing to expand the ability to analyze the serum proteome. In recent years, it has been realized that in addition to the circulating proteins, human serum also contains a large number of peptides. Many of these peptides are believed to be fragments of larger proteins that have been at least partially degraded by various enzymes such as metalloproteases. Identifying these peptides from a small amount of serum/plasma is difficult due to the complexity of the sample, the low levels of these peptides, and the difficulties in getting a protein identification from a single peptide. In this study, we modified previously published protocols for using centrifugal ultrafiltration, and unlike past studies did not digest the filtrate with trypsin with the intent of identifying endogenous peptides with this method. The filtrate fraction was concentrated and analyzed by a reversed phase-high performance liquid chromatography system connected to a nanospray ionization hybrid ion trap-Fourier transform mass spectrometer (LTQ-FTMS). The mass accuracy of this instrument allows confidence for identifying the protein precursors by a single peptide. The utility of this approach was demonstrated by the identification of over 300 unique peptides with 2 ppm or better mass accuracy per serum sample. With confident identifications, the origin and function of native serum peptides can be more seriously explored. Interestingly, over 34 peptide ladders were observed from over 17 serum proteins. This indicates that a cascade of proteolytic processes affects the serum peptidome. To examine whether this result was an artifact of serum, matched plasma and serum samples were analyzed with similar peptide ladders found in each.     

Applicability of continuous-flow fast atom bombardment liquid chromatography-mass spectrometry in bioanalysis. Dextromethorphan in plasma

Continuous-flow fast atom bombardment (CF-FAB) is an interface for combined liquid chromatography-mass spectrometry using FAB as the ionization method. The applicability of CF-FAB for quantitative bioanalysis was studied for a model compound, dextromethorphan, in plasma samples using conventional high-performance liquid chromatography. The flow-rate reduction was achieved either by splitting or by the phase-system switching approach. The features of both systems are discussed.     

高效液相色谱-串联质谱法测定肉制品和调味料中7种水产品过敏原

基于高效液相色谱-串联质谱系统建立了食品中水产品过敏原的快速筛查和定量检测方法。样品经蛋白质提取、纯化、胰蛋白酶解后，利用超高效液相色谱-四极杆/静电场轨道阱高分辨质谱（UPLC-Q/Exactive-HRMS）结合ProteinPilot软件，基于母离子和碎片离子谱分析，实现蛋白质和多肽的鉴定。再通过基本局部比对搜索工具（BLAST）与Uniprot数据库对比分析，筛选出南美白虾、大闸蟹、青蟹、金枪鱼、大西洋鲑鱼的7种过敏原蛋白的30个特征肽。利用高效液相色谱-三重四极杆质谱（UPLC-QqQ-MS）系统对特征肽进行验证和多反应监测（MRM）定量研究。结果表明，该方法在5~250 mg/kg范围内线性关系良好，检出限为2~3.5 mg/kg，平均回收率为88.7%~110.2%。该方法重现性好，通量高，可应用于肉制品和调味料中7种过敏原的快速筛查和定量分析。     

甲胺化衍生结合基于硅氢化物固定相的正相色谱用于单克隆抗体药物的N-糖基化表征

采用甲胺化衍生结合基于硅氢化物固定相的正相色谱（SiH-NPC）分析单抗的N-糖基化。样品经酶切、甲胺化衍生、纯化后由液相色谱-质谱进行分析。结果表明,相较于亲水相互作用色谱（HILIC）,SiH-NPC分离机制不同,使用常规的无盐流动相即可实现高分离度,避免污染质谱,色谱柱结构稳定,使用寿命长,更适合快速分析。结合唾液酸衍生方法,SiH-NPC在液相色谱-质谱联用鉴定酸性糖和糖异构体方面呈现显著优势,在生物制药行业中具有重要的应用潜力。     

Analysis of peptides and protein digests by reversed phase high performance liquid chromatography–electrospray ionisation mass spectrometry using neutral pH elution conditions

In this study, the advantages of carrying out the analysis of peptides and tryptic digests of proteins under gradient elution conditions at pH 6.5 by reversed-phase liquid chromatography (RP-HPLC) and in-line electrospray ionisation mass spectrometry (ESI-MS) are documented. For these RP separations, a double endcapped, bidentate anchored n-octadecyl wide pore silica adsorbent was employed in a capillary column format. Compared to the corresponding analysis of the same peptides and protein tryptic digests using low pH elution conditions for their RP-HPLC separation, this alternative approach provides improved selectivity and more efficient separation of these analytes, thus allowing a more sensitive identification of proteins at different abundance levels, i.e. more tryptic peptides from the same protein could be confidently identified, enabling higher sequence coverage of the protein to be obtained. This approach was further evaluated with very complex tryptic digests derived from a human plasma protein sample using an online two-dimensional (2D) strong cation-exchange (SCX)-RP-HPLC-ESI-MS/MS system. Again, at pH 6.5, with mobile phases of different compositions, improved chromatographic selectivities were obtained, concomitant with more sensitive on-line electrospray ionisation tandem mass spectrometric (ESI-MS/MS) analysis. As a consequence, more plasma proteins could be confidently identified, highlighting the potential of these RP-HPLC methods with elution at pH 6.5 to extend further the scope of proteomic investigations.     

Detection of nerve agent markers by liquid chromatography-mass spectrometry

An approach to the detection of metabolites of organophosphorous agents (OPA), such as O-iso-propylmethylphosphonic acid (detection limit, 4 ng/mL), O-pinacolylmethylphosphonic acid (0.6 ng/mL), and O-isobutylmethylphosphonic acid (1 ng/mL), in plasma samples was developed using liquid chromatography-mass spectrometry. The curves of the elutionxcretion of OPA metabolites were obtained for the samples of biological material of rats exposed to toxic substances. Determination was performed by mass spectrometry with electrospray ionization in the negative ion mode, using deprotonated molecules for detection. The biological samples were analyzed by reversed-phase chromatography using hydrophilic end-capped adsorbents. Solid phase extraction on reversed-phase adsorption cartridges containing a copolymer of styrene and divinylbenzene was proposed for sample preparation.     

A rapid method for cross-species quantitation of apolipoproteins A1, B48 and B100 in plasma by ultra-performance liquid chromatography/tandem mass spectrometry

Apolipoprotein B100 (apoB100) and apolipoprotein A1 (apoA1) are the primary protein components of low density lipoprotein (LDL) and high density lipoprotein (HDL) particles, respectively, and plasma levels of these proteins are associated with risks of cardiovascular disease. Existing apoB100 quantitation methods for animal models have been limited to affinity capture techniques such as enzyme-linked immunosorbent assay (ELISA) and Western blot which require specialized reagents for each species and in many cases are not readily available. Here we demonstrate a single translatable ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) assay that is fast and robust and can be used to measure apolipoprotein concentrations in plasma for six species. When possible, peptide sequences that are conserved across species were identified for this assay. The sample preparation is limited and can be carried out in 96-well microtiter plates and thus allows for multiplexed preparation of samples for analysis of large numbers of samples in a short time frame when combined with UPLC/MS/MS. Separation and quantitation of the tryptic peptides is carried out at 700 μL/min using a 1.7 μm core shell C18 column (2.1 × 50 mm). The chromatography is designed for the analysis of over 100 samples per day, and the UPLC run is less than 10 min. This assay is capable of supporting cardiovascular research by providing a single assay to measure critical biomarkers across multiple species without the need for antibodies, and does so in a high-throughput manner.     

Epitopic Peptides Identified by LC?CELISA and LC?CMS

A new strategy of a liquid chromatography (LC)-enzyme linked immunosorbent assay (ELISA) combined with liquid chromatography-mass spectrometry (LC?CMS) was developed to identify the epitopic peptides for the antigen in Streptococcus suis. Metallendopeptidase, a potential antigen for S. suis, was digested by trypsin. The tryptic solution was separated by LC under optimal condition and collected into tubes. After it was dried using a cooling vacuum, the immunogenecity of peptides in each tube was investigated by ELISA against its corresponding antibody. All peptides in each tube with positive signals based on ELISA results were identified by LC?CMS. Accordingly, six putative peptides of metallendopeptidase were found and synthesized, all of which possessed high antigenicity recognized by the antibody.     

Stress degradation study and structure characterization of oxidation degradation product of dexlansoprazole using liquid chromatography-mass spectrometry/time of flight,liquid chromatography-tandem mass spectrometry and nuclear magnetic resonance

The present study deals with the forced degradation behavior of dexlansoprazole under International Conference on Harmonisation (ICH) prescribed stress conditions. The drug was found to be more labile under acid, base, neutral, oxidative hydrolysis and thermal stress, while it was moderately stable under photolytic conditions. The known and unknown degradation products were separated on a C-18 column using a stability-indicating method. Liquid chromatography-mass spectrometry (LC-MS) analysis was performed for all the degradation studies. Isolation and structure characterization of oxidation degradation products were executed using sophisticated tools, viz. preparative high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry/time of flight (LC-MS/TOF), liquid chromatography-tandem mass spectrometry (LC-MS/MS), and nuclear magnetic resonance (NMR). This study demonstrates an ample methodology of degradation studies and structure elucidation of unknown degradation products of dexlansoprazole, which helps in the development and stability study of active pharmaceutical ingredients and formulated products.