Identification of Peptide tumor markers in a tumor graft model in immunodeficient mice

The medical demand for useful biomarkers is large and still increasing. This is especially true for cancer, because for this disease adequate diagnostic markers with high specificity and sensitivity are still lacking. Despite advances in imaging technologies for early detection of cancer, peptidomic multiplex techniques evolved in recent years will provide new opportunities for detection of low molecular weight (LMW) proteome biomarker (peptides) by mass spectrometry. Improvements in peptidomics research were made based on separation of peptides and/or proteins by their physico-chemical properties in combination with mass spectrometric detection, respectively identification, and sophisticated bioinformatic tools for data analysis. To evaluate the potential of serological tumor marker detection by differential peptide display (DPD) we analyzed plasma samples from a tumor graft model. After subcutaneous injection of HCT-116 cells in immunodeficient mice and their growth to a palpable tumor, plasma samples were analyzed by DPD. The comparison of obtained mass spectrometric data allows discovery of tumor specific peptides which fit well into the biological context of cancer pathogenesis and show a strong correlation to tumor growth. The identified peptides indicate events associated with hyper-proliferation and dedifferentiation of cells from an epithelial origin, which are typical characteristics of human carcinomas. We conclude that these findings are a "proof of principle" to detect differentially expressed, tumor-related peptides in plasma of tumor-bearing mice.     

Prerequisites for peptidomic analysis of blood samples: II. Analysis of human plasma after oral glucose challenge -- a proof of concept

Mass spectrometric plasma analysis for biomarker discovery has become an exploratory focus in proteomic research: the challenges of analyzing plasma samples by mass spectrometry have become apparent not only since the human proteome organization (HUPO) has put much emphasis on the human plasma proteome. This work demonstrates fundamental proteomic research to reveal sensitivity and quantification capabilities of our Peptidomics technologies by detecting distinct changes in plasma peptide composition in samples after challenging healthy volunteers with orally administered glucose. Differential Peptide Display (DPD) is a technique for peptidomics studies to compare peptides from distinct biological samples. Mass spectrometry (MS) is used as a qualitative and quantitative analysis tool without previous trypsin digestion or labeling of the samples. Circulating peptides (< 15 kDa) were extracted from 1.3 mL plasma samples and the extracts separated by liquid chromatography into 96 fractions. Each fraction was subjected to MALDI MS, and mass spectra of all fractions were combined resulting in a 2D-display of > 2,000 peptides from each sample. Endogenous peptides that responded to oral glucose challenge were detected by DPD of pre-and post-challenge plasma samples from 16 healthy volunteers and subsequently identified by nESI-qTOF MS. Two of the 15 MS peaks that were significantly modulated by glucose challenge were subsequently identified as insulin and C-peptide. These results were validated by using immunoassays for insulin and C-peptide. This paper serves as a proof of principle for proteomic biomarker discovery down to the pM concentration range by using small amounts of human plasma.     

Acid hydrolysis of proteins in matrix assisted laser desorption ionization matrices

Sample preparation is crucial to the success of experiments in biological mass spectrometry. In proteomics, digestion of the proteins into peptides is a key step for “bottom-up” approaches. Often, the use of enzymes requires physiological conditions, producing peptides that must be extracted or further purified before mass analysis. Chemical cleavage reagents offer more flexibility and can be more compatible with downstream mass analysis. Expanding on prior work using acid hydrolysis, proteolysis with matrix-assisted laser desorption ionization (MALDI) matrices is presented. This sample preparation can be performed rapidly with a minimum of reagents and sample handling, but it must first be evaluated in terms of digestion efficiency, missed cleavages, and side reactions before implementation for in-gel digestion and in-solution digestion using minimal volumes of protein. Time courses of acid hydrolysis are shown for protein standards, illustrating the sensitivity of this type of sample preparation, minimization of side reactions, and performance for proteins in mixtures. To illustrate the potential for sensitive detection of a specific protein, MALDI matrix hydrolysis is used to digest a protein immunoprecipitated from cell lysate.     

Peptidomics biomarker discovery in mouse models of obesity and type 2 diabetes

Type 2 diabetes mellitus (T2DM) is caused by the failure of the pancreatic beta-cell to secrete sufficient insulin to compensate a decreased response of peripheral tissues to insulin action. The pathological events causing beta-cell dysfunctions are only poorly understood and early markers that would predict islet function are missing. In contrast to immunoassays, unbiased proteomic technologies provide the opportunity to screen for novel marker protein and peptides of T2DM. An important subset of the proteome, peptides and peptide hormones secreted by the pancreas are deregulated in T2DM. The mass range of peptides and small proteins (1-20 kDa) is only sufficiently targeted by peptidomics, a combination of liquid chromatographic and mass spectrometric (MS) peptide analysis. Here, we describe the application of isotope label-free quantitative peptidomics to display and quantify relevant changes in the level of pancreatic peptides and peptide hormones in a preclinical model of T2DM, the Lep(ob)/Lep(ob) mouse. The amino acid sequence of statistical relevant top candidates was determined by MS/MS fragmentation or Edman degradation. The comparison of lean versus obese mice revealed increased levels of islet-specific peptides that can be divided into the following categories 1) the major islet peptide hormones insulin, amylin and glucagon; 2) proinsulin and C-peptide and 3) novel processing products of secretogranin, glucagon and amylin. Furthermore, we found increased levels of proteins and peptides implicated in zymogen granule maturation (syncollin) and nutritional digestion. In summary, our findings demonstrate that peptidomics is a valid approach to screen for novel peptide biomarkers.     

纳升液相色谱-质谱分析方法的重现性对尿液多肽组分析结果的影响

多肽组学是蛋白质组学技术的延伸和扩展,在医学和生物学研究中的应用日益广泛,但是,多肽组鉴定方法的重现性对实验结果的影响目前尚不清楚.本研究利用纳升液相色谱-高分辨质谱对健康人的尿液多肽组进行了7次平行分析,考察图谱数目、图谱利用率、鉴定的肽段数目、蛋白质数目、样品总离子强度和肽段保留时间等指标的变化,以揭示重复实验之间分析结果的可变性和稳定性.7次测定的肽段数目平均值为208,标准偏差为38;7次结果合并后,得到了归属于114个蛋白质的426个肽段,肽段和蛋白质数目均显著增加;而35个蛋白质的109个肽段在所有7次实验中均被检出,表明多肽组的单次分析结果既具有一定的随机性,又具有相对的稳定性.增加平行实验次数会扩大多肽组数据集,但测定3次以上后增加幅度减小.相比于肽段,多肽组的结果在蛋白质水平上更为稳定,提示利用蛋白质为多肽组的生物标志物更为稳健.     

The concept of functional peptidomics for the discovery of bioactive peptides in cell culture models

Detection and purification of novel bioactive peptides from biological sources is a scientific task that led to a substantial number of important discoveries. One major laborious approach used is the repetitive stepwise separation of the test sample into several fractions followed by the determination of their bioactivity, until purity allows for sequence identification. We tested whether functional peptidomics, a combination of biological read-outs with differential peptide display (DPD) is a suitable strategy to isolate bioactive peptides at lower workload and with improved success. Additionally, we evaluated the use of DPD to monitor the processing status of proinsulin by inhibition of the insulin processing pathway. The rat insulinoma cell line INS-1 stimulated either with 2 mmol/l or 10 mmol/l glucose was used as model to generate differential peptide displays. In parallel, the bioactivity of the supernatants from the INS-1 cells was measured by glucose uptake and lipolysis assays using the adipocyte cell line 3T3-L1. We were able to quickly and elegantly trace the known activity of insulin to increase glucose uptake and inhibit lipolysis. Following re-chromatography of selected fractions, relevant peptides were identified by DPD and bioassays: the rat insulin-1 precursor and two different insulin peptides. We demonstrated in a semi-quantitative fashion that inhibition of proinsulin processing leads to accumulation of the insulin precursor, and reduced secretion of insulin-1. Thus, we conclude that DPD is an attractive support technology in peptide purification strategies aiming to identify bioactive compounds, and is superior to ELISA in discriminating between the processing status of insulin and its precursor.     

A concise review of mass spectrometry imaging

Mass spectrometric imaging allows the investigation of the spatial distribution of molecules at complex surfaces. The combination of molecular speciation with local analysis renders a chemical microscope that can be used for the direct biomolecular characterization of histological tissue surfaces. MS based imaging advantageously allows label-free detection and mapping of a wide-range of biological compounds whose presence or absence can be the direct result of disease pathology. Successful detection of the analytes of interest at the desired spatial resolution requires careful attention to several steps in the mass spectrometry imaging protocol. This review will describe and discuss a selected number of crucial developments in ionization, instrumentation, and application of this innovative technology. The focus of this review is on the latest developments in imaging MS. Selected biological applications are employed to illustrate some of the novel features discussed. Two commonly used MS imaging techniques, secondary ion mass spectrometric (SIMS) imaging and matrix-assisted laser desorption ionization (MALDI) mass spectrometric imaging, center this review. New instrumental developments are discussed that extend spatial resolution, mass resolving power, mass accuracy, tandem-MS capabilities, and offer new gas-phase separation capabilities for both imaging techniques. It will be shown how the success of MS imaging is crucially dependent on sample preparation protocols as they dictate the nature and mass range of detected biomolecules that can be imaged. Finally, developments in data analysis strategies for large imaging datasets will be briefly discussed.     

Mass spectrometric imaging of peptide release from neuronal cells within microfluidic devices

Microfluidic devices are well suited for manipulating and measuring mass limited samples. Here we adapt a microfluidic device containing functionalized surfaces to chemically stimulate a small number of neurons (down to a single neuron), collect the release of neuropeptides, and characterize them using mass spectrometry. As only a small fraction of the peptides present in a neuron are released with physiologically relevant stimulations, the amount of material available for measurement is small, thereby requiring minimal sample loss and high-sensitivity detection. Although a number of detection schemes are used with microfluidic devices, mass spectrometric detection is used here because of its high information content, allowing the characterization of the released peptide complement. Rather than using an on-line approach, off-line analysis is used; after collection of the peptides onto a surface, mass spectrometric imaging interrogates that surface to determine the peptides released from the cell. The overall utility of this scheme is demonstrated using several device formats with measurement of neuropeptides released from Aplysia californica bag cell neurons.     

Detection of 25,000 molecules of Substance P by MALDI-TOF mass spectrometry and investigations into the fundamental limits of detection in MALDI

Mass spectrometric analysis of peptides with a total sample loading of several tens of thousands of molecules (i.e., low zeptomoles) is demonstrated. At this low level of sample loading, it becomes important to consider several very unique technical and fundamental aspects that are not obvious in compatible experiments with a higher amount of sample loading. We demonstrate that prudent matrix preparation allows analysis of peptides from solutions with picomolar concentrations in matrix-assisted laser desorption ionization (MALDI) mass spectrometry. Using microspot MALDI we demonstrate the introduction and detection of 25,000 molecules of Substance P in a time-of-flight mass spectrometer. A method based on probability theory is presented to estimate the minimum number of ions required for generating a statistically significant isotope peak pattern of peptide ions. It is found that the low boundary of ionization efficiency is 1–2% for Substance P. In addition, comparison of macro- and microspot sample deposition techniques for Substance P shows that under the experimental conditions used, a minimum of ∼5 analyte molecules per μm² are necessary to generate useful signals. Implications of these results on further mass spectrometric developments towards even more sensitive detection are discussed.     

纳升液相色谱-高分辨串联质谱研究冻存条件对唾液多肽组的影响

唾液多肽组学为疾病相关的生物标记物研究提供了新方法,但冻存条件对分析结果的影响并不清楚。本研究采用氧化石墨烯-磷酸镧纳米复合材料分离富集唾液多肽,利用纳升液相色谱-高分辨串联质谱技术,考察唾液样品分别置于-80℃与-20℃冻存6个月后对唾液多肽组的影响。结果表明,在-80℃冻存条件下的唾液样品中,共鉴定出归属于33种蛋白的429条肽段;在-20℃冻存条件下的唾液样品中,鉴定出595条肽段,对应31种蛋白。实验结果表明,相比于-80℃,唾液置于-20℃条件下的新增加肽段主要来源于已有肽段的降解,并且唾液中的蛋白质也发生了一定降解。本研究在肽段序列水平上考察了冻存条件对多肽的影响,结果表明,-20℃冻存条件不适合长期保存用于多肽组分析的唾液样品。本研究结果可为相关医学研究提供借鉴。     

Peptidomics: the integrated approach of MS, hyphenated techniques and bioinformatics for neuropeptide analysis

MS is currently one of the most important analytical techniques in biological and medical research. ESI and MALDI launched the field of MS into biology. The performance of mass spectrometers increased tremendously over the past decades. Other technological advances increased the analytical power of biological MS even more. First, the advent of the genome projects allowed an automated analysis of mass spectrometric data. Second, improved separation techniques, like nanoscale HPLC, are essential for MS analysis of biomolecules. The recent progress in bioinformatics is the third factor that accelerated the biochemical analysis of macromolecules. The first part of this review will introduce the basics of these techniques. The field that integrates all these techniques to identify endogenous peptides is called peptidomics and will be discussed in the last section. This integrated approach aims at identifying all the present peptides in a cell, organ or organism (the peptidome). Today, peptidomics is used by several fields of research. Special emphasis will be given to the identification of neuropeptides, a class of short proteins that fulfil several important intercellular signalling functions in every animal. MS imaging techniques and biomarker discovery will also be discussed briefly.     

Recent developments and applications of capillary and microchip electrophoresis in proteomics and peptidomics (2015–mid 2018)

This review summarizes recent developments and applications of capillary and microchip electroseparation methods in proteomic and peptidomic analyses since the year 2015 to ca. mid 2018. Sample preparation procedures for the removal of interfering components or for pre‐fractionation and preconcentration of proteins and peptides of interest are discussed. The innovations in coupling of capillary or microchip electroseparation methods with different modes of mass spectrometry detection are covered. In addition, significant recent applications of capillary electromigration methods in both bottom‐up and top‐down proteomics as well as in determinations of post‐translational modifications of proteins are presented. Moreover, several examples of the utilization of capillary electromigration methods coupled with mass spectrometry detection for clinical proteomics and peptidomics are described.     

Validation of a bioanalytical method for the quantification of a therapeutic peptide, ramoplanin, in human dried blood spots using LC-MS/MS

A method has been developed and validated for the quantification of ramoplanin, a 2554 Da peptide antibiotic, in human dried blood spots using high‐performance liquid chromatography with tandem mass spectrometric detection. The validation data meet FDA acceptance criteria for bioanalytical assays and cover the quantification of ramoplanin over the range 10–5000 ng/mL. The assay determines ramoplanin at the same lower limit of quantification as conventional liquid sample methods. Dried blood spot analysis provides an approach for quantification of peptide therapeutics and delivers significant benefits for sample collection and handling and also sample cleanup over conventional plasma and serum assays. Copyright © 2010 John Wiley & Sons, Ltd.     

On-line isotachophoretic sample focusing for loadability enhancement in capillary electrochromatography-mass spectrometry

The use of isotachophoretic (ITP) sample focusing to improve the detection limits for the analysis of charged compounds in capillary electrochromatography (CEC) is described. A coupled-column set-up was used with a 220-microm inner diameter capillary, in which counterflow ITP focusing was performed, connected via a T-junction to a 75-microm inner diameter CEC capillary. As is illustrated, the use of ITP focusing resulted in a dramatic reduction of the sample concentration detection limits. To demonstrate the performance of the ITP-CEC combination, several cationic low-molecular mass compounds in a plasma and urine matrix are analysed using UV-absorbance and mass spectrometric detection. A linear calibration curve was constructed over three decades and detection limits in the low nmol/l range were found for academic samples, using UV-absorbance detection.     

Discovery of lung squamous carcinoma biomarkers by profiling the plasma peptide with LC/MS/MS

Biomarkers can be used for the screening and clinical diagnosis of cancer, and peptidomics approach has been proven successful in the research of biomarkers. To develop better peptidomic technologies for fast, accurate, and reliable detection of peptides biomarkers for lung cancer, we have improved the procedures of blood collection to minimize the degradation of the blood proteins and optimize the extraction of peptidome peptides from plasma samples based on acetonitrile precipitation associated with size exclusion chromatography (SEC). Studies show that squamous cell carcinomas are found to express CAGE1, SPAT9 and TEX28 genes at significantly higher rates, and the results suggest that as tumors progress, the level of CAGE1, SPAT9 and TEX28 genes are likely to increase and lead to immunization. This suggests a potentially important therapeutic method for cancer testis-based cancer vaccines.     

Operational variables in high-performance liquid chromatography-electrospray ionization mass spectrometry of peptides and proteins using poly(styrene-divinylbenzene) monoliths

Capillary reversed-phase high-performance liquid chromatography (RP-HPLC) utilizing monolithic poly(styrene-divinylbenzene) columns was optimized for the coupling to electrospray ionization mass spectrometry (ESI-MS) by the application of various temperatures and mobile phase additives during peptide and protein analysis. Peak widths at half height improved significantly upon increasing the temperature and ranged from 2.0 to 5.4 s for peptide and protein separations at 70 degrees. Selectivity of peptide elution was significantly modulated by temperature, whereas the effect on proteins was only minor. A comparison of 0.10% formic acid (FA), 0.050% trifluoroacetic acid (TFA), and 0.050% heptafluorobutyric acid (HFBA) as mobile phase additives revealed that highest chromatographic efficiency but poorest mass spectrometric detectabilities were achieved with HFBA. Clusters of HFBA, water, and acetonitrile were observed in the mass spectra at m/z values >500. Although the signal-to-noise ratios for the individual peptides diverged considerably both in the selected ion chromatograms and extracted mass spectra, the average mass spectrometric detectabilities varied only by a factor of less than 1.7 measured with the different additives. Limits of detection for peptides with 500 nl sample volumes injected onto a 60 mm x 0.20 mm monolithic column were in the 0.2-13 fmol range. In the analysis of hydrophobic membrane proteins, HFBA enabled highest separation selectivity at the cost of lower mass spectral quality. The use of 0.050% TFA as mobile phase additive turned out to be the best compromise between chromatographic and mass spectrometric performance in the analysis of peptides and proteins by RP-HPLC-ESI-MS using monolithic separation columns.     

Identification of novel biomarker candidates by differential peptidomics analysis of cerebrospinal fluid in Alzheimer's disease

The objective of this work was the application of peptidomics technologies for the detection and identification of reliable and robust biomarkers for Alzheimer's disease (AD) contributing to facilitate and further improve the diagnosis of AD. Using a new method for the comprehensive and comparative profiling of peptides, the differential peptide display (DPD), 312 cerebrospinal fluid (CSF) samples from AD patients, cognitively unimpaired subjects and from patients suffering from other primary dementia disorders were analysed as four independent analytical sets. By combination with a cross validation procedure, candidates were selected from a total of more than 6,000 different peptide signals based on their discriminating power. Twelve candidates were identified using mass-spectrometric techniques as fragments of the possibly neuroprotective neuroendocrine protein VGF and another one as the complement factor C3 descendent C3f. The combination of peptide profiling and cross validation resulted in the detection of novel potential biomarkers with remarkable robustness and a close relation to AD pathophysiology.     

Use of nonspecific cleavage products for protein sequence analysis as shown on calcyclin isolated from human granulocytes

In this paper, analysis strategies developed for a sequencing problem concerning the identification of an S100 protein isolated from human granulocytes are discussed. The analysis of a trypsinized lyophilized sample suggested the presence of a number of peptides which are non-tryptic in origin. During purification of proteins from cell lysates nonspecific cleavage can be observed which may reflect biological processes and can become an unavoidable analytical problem. Current mass spectrometric software is evaluated for the analysis of nonspecific digests in this context. Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), high-performance liquid chromatography (HPLC)-MS/MS, and selected ion monitoring (SIM)-MS/MS have been used for peptide analysis and in addition HPLC-MS was carried out for protein analysis leading to the detection of an N-terminal modification of the protein. The success of the study is mainly due to the careful investigation of nonspecific cleavage products. Data obtained from the routine mass spectrometric analysis of an in-gel-digest allowed the identification of this protein as S100 calcium-binding protein A6-calcyclin whose expression in granulocytes has not been described so far.     

Determination of Methylphosphonic Acid in Human Blood Plasma by High-Performance Liquid Chromatography–Tandem Mass Spectrometry

Using high-performance liquid chromatography combined with tandem mass spectrometric detection, an approach has been developed for the determination of the most stable nerve agent biomarker, methylphosphonic acid, in human blood plasma. The proposed method is based on the derivatization of methylphosphonic acid with p-bromophenacyl bromide. The optimization of conditions for human plasma sample preparation, mass spectrometric detection conditions, and gradient elution program has been performed. The proposed approach has demonstrated satisfactory reproducibility and selectivity of the determination; the limit of detection for methylphosphonic acid in human plasma was 3 ng mL^–1.     

India ink staining after sodium dodecyl sulfate polyacrylamide gel electrophoresis and in conjunction with Western blots for peptide mapping by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

We present an approach that allows matrix-assisted laser desorption/ionization time-of-flight mass spectrometric (MALDI-TOFMS) peptide mapping of proteins separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto nitrocellulose (NC). After blocking the nitrocellulose membrane with polyvinylpyrrolidone-40 the immobilized proteins are visualized using India Ink staining which allows the detection of low nanogram amounts of protein. The utilization of a low concentration of Tween 20 (0.05%) in the India Ink staining solution does not negatively impair the quality of the mass spectra. Due to the virtual nondestructive nature of the stain proteolytic peptides could be recovered from the NC membrane. Taking into account minor precautions during the sample manipulation and concentration and by loading the sample onto a pre-crystallized matrix layer, high quality mass spectral data were obtained on <100 femtomoles of protein loaded onto the gel. Finally, the use of India Ink in conjunction with Western blot analysis is also demonstrated. A rat plasma protein, characterized by Western blot as a covalently modified protein-drug compound, was subjected to peptide mapping and post source decay (PSD) sequencing of peptides. The zomepirac-modified protein was identified as the alpha-subunit of fibrinogen.