Usefulness of an integrated microfluidic device (HPLC-Chip-MS) to enhance confidence in protein identification by proteomics

Nanoflow liquid chromatography/mass spectrometry (nanoLC/MS) has become a current tool in proteomics applications increasingly used in the search for new biomarkers. A new integrated microfluidic device (HPLC-Chip), coupled to ion trap mass spectrometry (ITMS), appears as an innovative and robust tool for improving the identifications commonly performed by nanoLC/MS/MS. We tested this device for the identification of proteins obtained from two-dimensional gel electrophoresis or chromatography. The chip allows the measurement of reproducible retention times that, in association with m/z ratios, was found useful for identifying peptide sequences without ambiguity. A sensitivity increase of a factor of at least 5-fold is obtained compared to the results obtained previously in our laboratory by conventional nanoLC/MS/MS on the same ion trap. We conclude that this recently available microfluidic device can be a valuable tool during biomarker discovery programs, particularly identifying low-abundance proteins.     

Improving peptide identification using an empirical peptide retention time database

Peptide retention time (RT) is independent of tandem mass spectrometry (MS/MS) parameters and can be combined with MS/MS information to enhance peptide identification. In this paper, we utilized peptide empirical RT and MS/MS for peptide identification. This new approach resulted in the construction of an Empirical Peptide Retention Time Database (EPRTD) based on peptides showing a false‐positive rate (FPR) ≤1%, detected in several liquid chromatography (LC)/MS/MS analyses. In subsequent experiments, the RT of peptides with FPR >1% was compared with empirical data derived from the EPRTD. If the experimental RT was within a specified time range of the empirical value, the corresponding MS/MS spectra were accepted as positive. Application of the EPRTD approach to simple samples (known protein mixtures) and complex samples (human urinary proteome) revealed that this method could significantly enhance peptide identification without compromising the associated confidence levels. Further analysis indicated that the EPRTD approach could improve low‐abundance peptides and with the expansion of the EPRTD the number of peptide identifications will be increased. This approach is suitable for large‐scale clinical proteomics research, in which tens of LC/MS/MS analyses are run for different samples with similar components. Copyright © 2008 John Wiley & Sons, Ltd.     

Hydrophilic properties as a new contribution for computer-aided identification of short peptides in complex mixtures

A new method to predict elementary amino acid (AA) composition of peptides (molar mass <1,000 g/mol) is described. This procedure is based on a computer-aided method using three combined analyses-reversed phase liquid chromatography (RPLC), hydrophilic interaction chromatography (HILIC) and capillary electrophoresis coupled with mass spectrometry-and using a software calculating all possible amino acid combinations from the mass of any given peptide. The complementarity between HILIC and RPLC was demonstrated. Peptide retention prediction in HILIC was successfully modelled, and the achieved prediction accuracy was as high as r2=0.97. This mathematical model, based on amino acid retention contributions and peptide length, provided the information about peptide hydrophilicity that was not redundant with its hydrophobicity. Correlations between respectively the hydrophobicity coefficients and RPLC retention time, hydrophilicity and HILIC retention time, and electrophoretic mobility and migration time were used for ranking all potential AA combinations corresponding to the given mass. The essential contribution of HILIC in this identification strategy and the need to combine the three models to significantly increase identification capabilities were both shown. Applied to an 18-standard peptide mixture, the identification procedure enabled the actual AA combination determination of the 14 di- to pentapeptides, in addition to an over 98 % reduction of possible combination numbers for the four hexapeptides. This procedure was then applied to the identification of 24 unknown peptides in a rapeseed protein hydrolysate. The effective AA composition was found for ten peptides, whereas for the 14 other peptides, the number of possible combinations was reduced by over 95 % thanks to the association of the three analyses. Finally, as a result of the information provided by the analytical techniques about peptides present in the mixture, the proposed method could become a highly valuable tool to recover bioactive peptides from undefined protein hydrolysates.     

蛋白质组质谱分析中基于串并联支持向量机的肽段色谱保留时间预测方法

基于质谱的大规模蛋白质鉴定中,在线液相色谱分离发挥了重要作用。色谱保留时间(retention time,RT)是肽段鉴定和定量的重要信息。由于整个色谱分析运行时间中,流动相中的有机相采用了非线性浓度曲线以及样品中肽段之间的相互影响等因素,基于肽段序列的RT预测还存在精度不高、模型推广性能差等问题。本文提出了一种基于串并联支持向量机(serial and parallel support vector machine,SP-SVM)的RT预测方法,能够表征洗脱过程中有机相浓度的非线性变化和肽段之间的相互影响,显著提高了肽段保留时间预测的精度。利用复杂样本数据集验证结果表明,预测RT和实验RT之间的决定系数达到了0.95,超过95%的鉴定肽段的RT预测误差范围小于总运行时间的20%,超过70%的鉴定肽段的RT预测误差范围小于总运行时间的10%。本文提出的模型的性能达到了目前已知的最好水平。     

基于深度学习的保留时间预测方法的研究进展及应用

在基于液相色谱-质谱联用的蛋白质组学研究中,肽段的保留时间作为有效区分不同肽段的特征参数,可以根据肽段自身的序列等信息对其进行预测.使用预测得到的保留时间辅助质谱数据鉴定肽段序列可以提高鉴定的准确性,因此对保留时间预测的工作一直受到领域内的广泛关注.传统的保留时间预测方法通常是根据氨基酸序列计算肽段的理化性质,进而计算...     

Poly(dimethylsiloxane)-based microfluidic device with electrospray ionization-mass spectrometry interface for protein identification

An easy method to fabricate poly(dimethylsiloxane) (PDMS)-based microfluidic chips for protein identification by tandem mass spectrometry is presented. This microchip has typical electrophoretic microchannels, a flow-through sampling inlet, and a sheathless nanoelectrospray ionization (ESI) interface. The surface of the microchannel was modified with 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) and the generated electroosmotic flow under acidic buffer condition used for the separation was found to be more stable compared to that generated by the microchannel without modification. The feasibility of the device for flow-through sampling, separation, and ESI-MS/MS analysis was demonstrated by the analysis of a standard mixture composed of three tryptic peptides. Results show that four peaks corresponding to three peptide standards and acetylated products of the standard peptide were well resolved and the deduced sequences were consistent with those expected. Furthermore, the compatibility of this device with other miniaturized devices to integrate the whole process was also explored by connecting a miniaturized enzymatic digestion cartridge and a desalting cartridge in series to the sampling inlet of the microchip for the identification of a model protein, beta-casein.     

Alternative methods for verifying the results of the mass spectrometric identification of peptides in shotgun proteomics

Database search is the most popular approach used for the identification of peptides in contemporary shotgun proteomics; it utilizes only mass spectrometric data. In this work, we introduce three criteria for the verification of peptide identification; these are based on the analysis of data orthogonal to tandem mass spectra. The first one utilizes chromatographic retention times of peptides. The development of such approaches has been hindered by the relatively low accuracy of retention time prediction algorithms. In this work, we suggest the use of two independent models of the liquid chromatography of peptides, which increase the reliability of the results. The second criterion utilizes the mean number of missed tryptic cleavages per peptide. The third one results from the analysis of the difference between theoretical and experimentally measured peptide masses. The proposed criteria were applied to the tandem mass spectra of tryptic peptides from rat kidney tissue, which were processed by the Mascot search engine. All the criteria showed that Mascot significantly overestimated the reliability of an identification. This conclusion was supported by the PeptideProphet algorithm.     

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.     

Integrated electrokinetically driven microfluidic devices with pH‐mediated solid‐phase extraction coupled to microchip electrophoresis for preterm birth biomarkers

Integration in microfluidics is important for achieving automation. Sample preconcentration integrated with separation in a microfluidic setup can have a substantial impact on rapid analysis of low‐abundance disease biomarkers. Here, we have developed a microfluidic device that uses pH‐mediated solid‐phase extraction (SPE) for the enrichment and elution of preterm birth (PTB) biomarkers. Furthermore, this SPE module was integrated with microchip electrophoresis for combined enrichment and separation of multiple analytes, including a PTB peptide biomarker (P1). A reversed‐phase octyl methacrylate monolith was polymerized as the SPE medium in polyethylene glycol diacrylate modified cyclic olefin copolymer microfluidic channels. Eluent for pH‐mediated SPE of PTB biomarkers on the monolith was optimized using different pH values and ionic concentrations. Nearly 50‐fold enrichment was observed in single channel SPE devices for a low nanomolar solution of P1, with great elution time reproducibility (<7% RSD). The monolith binding capacity was determined to be 400 pg (0.2 pmol). A mixture of a model peptide (FA) and a PTB biomarker (P1) was extracted, eluted, injected, and then separated by microchip electrophoresis in our integrated device with ∼15‐fold enrichment. This device shows important progress towards an integrated electrokinetically operated platform for preconcentration and separation of biomarkers.     

基于保留时间和质荷比匹配的液相色谱-质谱联用技术用于非标记肽段的差异分析

建立了一种无需化学标记的,基于纳升级毛细管液相色谱-电喷雾离子阱质谱联用技术和质谱数据处理的肽段差异分析方法。本方法采用定量差异分析与肽序列鉴定分析分别进行的策略,首先对样品进行质谱全扫描的液质全谱式分析,在全扫描质谱数据中提取肽特征点信息,通过保留时间和质荷比参数匹配不同样品中的共有肽特征点,比较其相对峰强度有无差异。最后对样品中存在丰度差异的肽特征点进行选择性二级质谱分析和序列鉴定,从而实现复杂样品中肽段的差异比较分析。以血浆蛋白酶解混合物为实验对象,考察了本方法用于肽段相对定量分析的重现性以及浓度信号响应曲线等。结果表明:提取的肽特征点峰强度相对标准偏差的中值<22%,肽段离子强度动态范围达3个数量级,在5～1000fmol范围内对肽段定量具有良好线性关系。本方法可用于不同条件样品中具有倍数差异的内源性肽的比较分析。     

Epitope mapping of allergen ovalbumin using biofunctionalized magnetic beads packed in microfluidic channels The first step towards epitope-based vaccines

Specific allergen immunotherapy is frequently associated with adverse reactions. Several strategies are being developed to reduce the allergenicity while maintaining the therapeutic benefits. Peptide immunotherapy is one such approach. Methods for the simple and rapid identification of immunogenic epitopes of allergens (i.e. allergenic epitopes) are ongoing and could potentially lead to peptide-based vaccines. An epitope extraction technique, based on biofunctionalized magnetic microspheres self-organized under a magnetic field in a channel of a simple microfluidic device fabricated from polydimethylsiloxane, was applied in the isolation and identification of prospective allergenic epitopes. Similarly to chromatographic column separations, the easily replaceable plug of self-organized beads in the channel benefits especially from an even larger surface-to-volume ratio and an enhanced interaction of the surfaces with passing samples. Ovalbumin, the major protein of egg white and a typical representative of food allergens, was selected as the model molecule. Highly resistant ovalbumin was at first efficiently digested by a magnetic proteolytic reactor with trypsin treated with l-1-tosylamido-2-phenylethyl chloromethyl ketone and the second step, i.e. capture of allergenic epitopes from the mixture of peptides, was performed by a magnetic immunoaffinity carrier with orientedly immobilized rabbit anti-ovalbumin IgG molecules. Captured peptides were released with 0.05% trifluoroacetic acid. The elution fractions were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The peptide fragment of ovalbumin HIATNAVLFFGR (m/z: 1345.75, position: 371-382) was identified as a relevant allergenic epitope in this way. Such a microfluidic magnetic force-based epitope extraction technique applied in the epitope mapping of ovalbumin has the potential to be a significant step towards developing safe and cost-effective epitope-based vaccines.     

Integrated microfluidic devices with enhanced separation performance: application to phosphoproteome analyses of differentiated cell model systems

This work reports on the application of a microfluidic device integrating nanoscale LC to nanoelectrospray MS (nano-LC-chip-MS) for the analysis of complex protein digests. Peak profile analyses of more than 700 peptide ions, reproducibly detected across replicate nano-LC-chip-MS runs (n = 5), indicated that the system provided RSD values of 0.24% on retention time, +/- 30 ppm on m/z measurement and +/- 30% variation on intensity over three orders of magnitude. RP adsorbant media with different alkyl chains and particle size packed in both trapping and separation channels were investigated to improve the chromatographic performance of this system. A two-fold improvement in chromatographic peak capacity was achieved using microfluidic devices comprising a 5 mircrom C3 trap with 2.5 microm C18 trap separation channel compared to the traditional 5 microm C18 stationary phase. Enhanced sample selectivity for the identification of phosphopeptides was obtained by combining immobilized metal affinity media prior to peptide separation on the RP microfluidic device. This system was evaluated in the context of differential phosphoproteome analyses to identify changes in signaling events and protein expression of human monocytes following the administration of phorbol ester.     

Multi-dimensional HPLC/MS of the nucleolar proteome using HPLC-chip/MS

The proteome of the human nucleolus was investigated in a single analysis using off-line strong cation exchange chromatography and microfraction collection combined with HPLC-chip/MS. The analysis was conducted either as a 1-D workflow with HPLC-chip alone or as a 2-D workflow. Two hundred and six unique proteins were identified in the International Protein Index human database corresponding to 2024 unique tryptic peptides identified in the 2-D analysis. In contrast, only 34 proteins and 151 corresponding tryptic peptides were found by applying a 1-D separation strategy. This clearly indicated that the complexity of the samples required the combination of more than one orthogonal separation technique. Stringent database search criteria, including reversal of sequences and therefore better exclusion of false-positive identifications, were applied for reliable protein identification.     

MSQ: a tool for quantification of proteomics data generated by a liquid chromatography/matrix‐assisted laser desorption/ionization time‐of‐flight tandem mass spectrometry based targeted quantitative proteomics platform

Mass spectrometry (MS)‐based quantitative proteomics has become a critical component of biological and clinical research for identification of biomarkers that can be used for early detection of diseases. In particular, MS‐based targeted quantitative proteomics has been recently developed for the detection and validation of biomarker candidates in complex biological samples. In such approaches, synthetic reference peptides that are the stable isotope labeled version of proteotypic peptides of proteins to be quantitated are used as internal standards enabling specific identification and absolute quantification of targeted peptides. The quantification of targeted peptides is achieved using the intensity ratio of a native peptide to the corresponding reference peptide whose spike‐in amount is known. However, a manual calculation of the ratios can be time‐consuming and labor‐intensive, especially when the number of peptides to be tested is large. To establish a liquid chromatography/matrix‐assisted laser desorption/ionization time‐of‐flight tandem mass spectrometry (LC/MALDI TOF/TOF)‐based targeted quantitative proteomics pipeline, we have developed a software named Mass Spectrometry based Quantification (MSQ). This software can be used to automate the quantification and identification of targeted peptides/proteins by the MALDI TOF/TOF platform. MSQ was applied to the detection of a selected group of targeted peptides in pooled human cerebrospinal spinal fluid (CSF) from patients with Alzheimer's disease (AD) in comparison with age‐matched control (OC). The results for the automated quantification and identification of targeted peptides/proteins in CSF were in good agreement with results calculated manually. Copyright © 2010 John Wiley & Sons, Ltd.     

The characterization of column heating effect in nanoflow liquid chromatography mass spectrometry (nanoLC‐MS)–based proteomics

Column heating strategy is often applied in nano–high‐performance liquid chromatography–mass spectrometer (nanoHPLC‐MS) platform for enhancing the analytical efficiency of peptides or proteins. Nonetheless, the influence effects of column heating in peptides or proteins identification still lack of deep understanding. In this study, a systematic comparison of room temperature (RT) and column heating of nanoHPLC was done. Based on the data, under column heating condition, the backpressure of nanoHPLC can be decreased. Due to the increase of resolution, the peak widths of precursor ion were narrowed. As a result, in MS/MS data acquisition part, more time was spared for MS1 detecting and MS2 fragmenting, which eventually resulted in increased identification of peptides and proteins. Moreover, we also proposed the application scope of column heating by evaluating its influence on sample detection. On one hand, column heating significantly increased the identification of membrane proteins due to more efficient elution of highly hydrophobic peptides compared with RT. On the other hand, heating was not suitable for analyzing short or/and hydrophilic peptides with low retention time, which would be eluted out during sample loading process under high temperature and missed by mass spectrometric detection. In conclusion, our study provides a reference for rational application of column heating in proteomics research.     

色谱保留时间在蛋白质组研究中的应用

液相色谱与串联质谱联用(LC-MS/MS)技术是蛋白质组学研究中的常见方法。保留时间作为独立于质谱信息的参数已经被用于蛋白质的鉴定和定量工作中。在多肽鉴定领域,多肽的色谱保留时间预测与常规的二级串联质谱数据库搜索算法结合可以提高鉴定的可信度。鉴定的灵敏度也可以通过匹配多次LC-MS实验中具有相同精确质量数和保留时间的峰而提高。另一方面,由于色谱条件的微小改变即会引起保留时间的变化,因此对多次实验结果进行保留时间比对是进行非标记定量的不可或缺的步骤。另外,联合保留时间偏移和质量数信息还可以进行蛋白质翻译后修饰(post-translational modification, PTM)的鉴定。     

Assessment of acetone as an alternative to acetonitrile in peptide analysis by liquid chromatography/mass spectrometry

Acetonitrile as a solvent used in liquid chromatography/mass spectrometry (LC/MS) of peptides and proteins is a relatively toxic solvent (LD₅₀ oral; rat; 2,460 mg/kg) compared to alternatives like methanol (LD₅₀ oral; rat; 5,628 mg/kg) and acetone (LD₅₀ oral; rat; 5,800 mg/kg). Strategies to minimize its consumption in LC are either to reduce the inner diameter of the column or replace acetonitrile with a suitable alternative. Methanol is often recommended to replace acetonitrile in peptide analysis. In this study however, the main focus lies on another alternative solvent for LC/MS of peptides; acetone. A number of model proteins were tryptically digested and the peptide solutions were analyzed on a linear trap quadrupole (LTQ) mass spectrometer. The performances of acetonitrile, methanol and acetone were compared according to the quality of the chromatograms obtained and identification of the peptides using the BioWorks? software developed by Thermo Scientific. In accordance to the elutropic series, acetone was found to significantly reduce the retention times of peptides separated by C18 column material with regard to acetonitrile while methanol led to increased retention times. Acetone was the superior solvent to methanol for most of the tested model proteins reaching similar sequence coverage and numbers of identified peptides as acetonitrile. We therefore propose acetone as an alternative to acetonitrile in LC/MS of peptides. Copyright © 2009 John Wiley & Sons, Ltd.     

Retention Characteristics of Peptides in RP-LC: Peptide Retention Prediction

A review of recent results of the use of chromatographic retention data in peptide identification and in the development of procedures for peptide retention prediction is presented. In recent years, reversed phase LC (RP-LC) has become an important tool in the separation of peptides in MS analysis. A challenging problem in a further expansion of RP-LC applications is the use of already available retention information for the identification purposes simultaneously with MS–MS identification. This overview focuses on the retention characteristics suggested in LC. We will discuss the application of the retention index concept in LC, which is widely used in GC to characterize retention of organic compounds. The use of retention indices as retention characteristics of analytes in LC was first suggested at the end of 1970s, however the application of retention indices is still somewhat rare today. There are several reasons for this. One is the relatively high sensitivity and variability of retention indices to the change of parameters of chromatographic systems. Another is the chemical restrictions in the search of the universal set of reference compounds suitable for retention scaling. Several methods were suggested for the prediction of the retention times of peptides. A frequently used approach is based on the additivity scheme and calculation of the elution time through the summation of retention coefficients of amino acids constituting the peptide. Such an approach allows fairly accurate predictions of the retention time of peptides made up of not more then 15–20 amino acid residues. Additional correction factors were suggested to improve predictions including corrections for the peptide length, peptide hydrophobicity, sequence of amino acids, etc. Suggested procedures are discussed in detail. Application of predicted retention times in the identification of peptides is considered. Current status of LC retention data collections is presented.     

A method for calculating <Superscript>16</Superscript>o/<Superscript>18</Superscript>o peptide ion ratios for the relative quantification of proteomes

A method is described for the identification and relative quantification of proteomes using accurate mass tags (AMT) generated by nLC-dual ESI-FT-ICR-MS on a 7T instrument in conjunction with stable isotope labeling using 16O/18O ratios. AMTs were used for putative peptide identification, followed by confirmation of peptide identity by tandem mass spectrometry. For a combined set of 58 tryptic peptides from bovine serum albumin (BSA) and human transferrin, a mean mass measurement accuracy of 1.9 ppm +/-0.94 ppm (CIM99%) was obtained. This subset of tryptic peptides was used to measure 16O/18O ratios of 0.36 +/- 0.09 (CIM99%) for BSA (micro = 0.33) and 1.48 +/- 0.47 (CIM99%) for transferrin (micro = 1.0) using a method for calculating 16O/18O ratios from overlapping isotopic multiplets arising from mixtures of 16O, 18O1, and 18O2 labeled C-termini. The model amino acid averagine was used to calculate a representative molecular formula for estimating and subtracting the contributions of naturally occurring isotopes solely as a function of peptide molecular weight. The method was tested against simulated composite 16O/18O spectra where peptide molecular weight, 16O/18O ratio, 18O1/18O2 ratios, and number of sulfur atoms were varied. Relative errors of 20% or less were incurred when the 16O/18O ratios were less than three, even for peptides where the number of sulfur atoms was over- or under-estimated. These data demonstrate that for biomarker discovery, it is advantageous to label the proteome representing the disease state with 18O; and the method is not sensitive to variations in 18O1/18O2 ratio. This approach allows a comprehensive differentiation of expression levels and tentative identification via AMTs, followed by targeted analysis of over- and under-expressed peptides using tandem mass spectrometry, for applications such as the discovery of disease biomarkers.     

The Utility of Accurate Mass and LC Elution Time Information in the Analysis of Complex Proteomes

The combination of mass and normalized elution time (NET) of a peptide identified by liquid chromatography-mass spectrometry (LC-MS) measurements can serve as a unique signature for that peptide. However, the specificity of an LC-MS measurement depends upon the complexity of the proteome (i.e., the number of possible peptides) and the accuracy of the LC-MS measurements. In this work, theoretical tryptic digests of all predicted proteins from the genomes of three organisms of varying complexity were evaluated for specificity. Accuracy of the LC-MS measurement of mass-NET pairs (on a 0 to 1.0 NET scale) was described by bivariate normal sampling distributions centered on the peptide signatures. Measurement accuracy (i.e., mass and NET standard deviations of +/-0.1, 1, 5, and 10 ppm, and +/-0.01 and 0.05, respectively) was varied to evaluate improvements in process quality. The spatially localized confidence score, a conditional probability of peptide uniqueness, formed the basis for the peptide identification. Application of this approach to organisms with comparatively small proteomes, such as Deinococcus radiodurans, shows that modest mass and elution time accuracies are generally adequate for confidently identifying most peptides. For more complex proteomes, more accurate measurements are required. However, the study suggests that the majority of proteins for even the human proteome should be identifiable with reasonable confidence by using LC-MS measurements with mass accuracies within +/-1 ppm and high efficiency separations having elution time measurements within +/-0.01 NET.