Label-free molecular interaction determinations with nanoscale interferometry

Quantification of protein-protein and ligand-substrate interactions is central to understanding basic cellular function and for evaluating therapeutics. To mimic biological conditions, such studies are best executed without modifying the proteins or ligands (i.e., label-free). While tools for label-free assays exist, they have limitations making them difficult to fully integrate into microfluidic devices. Furthermore, it has been problematic to reduce detection volumes for on-channel universal analyte quantification without compromising sensitivity, as needed in label-free methods. Here we show how backscattering interferometry in rectangular channels (BIRC) facilitates label-free studies within picoliter volumes. The simple and unique optical train was based on rectangular microfluidic channels molded in poly(dimethylsiloxane) and low-power coherent radiation. Quantification of irreversible streptavidin-biotin binding and reversible protein A-human IgG Fc molecular interactions in a 225 pL detection volume was carried out label-free and noninvasively. Detection limits of 47 x 10(-15) mol of biotin reacted with surface-immobilized streptavidin were achieved. In the case of reversible interactions of protein A and the Fc fragment of human IgG, detection limits were determined to be 2 x 10(-15) mol of IgG Fc. These experiments demonstrate for the first time that (1) high-sensitivity universal solute quantification is possible using interferometry performed within micrometer-sized channels formed in inexpensive PDMS chips, (2) label-free reversible molecular interaction can be studied with femtomoles of solute, and (3) BIRC has the potential to quantify binding affinities in a high-throughput format.     

Exponential isothermal amplification coupled MALDI-TOF MS for microRNAs detection

MicroRNAs (miRNAs) have attracted significant attention in biomedical research and clinical diagnosis. However, due to their inherent characteristics of low abundance and the high complexity of corresponding biological matrices, simultaneous detection of multiple miRNAs at low abundance is still a challenge. In this work, a method coupling exponential amplification reaction (EXPAR) with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is developed for label-free and simultaneous detection of multiple miRNAs. The assay can be performed under isothermal conditions in a single reaction tube, and finished in less than 30 min. It exhibits good quantification ability and with attomolar-level sensitivity for miRNAs detection. It also shows high specificity to distinguish miRNAs at single-nucleotide resolution. We used the method to detect the miRNA-21, let-7a, miRNA-100, and miRNA-125b in samples of spiked human serum and breast cancer cells (i.e., MCF-7, MDA-MB-231 and SK-BR-3). The quantification results were well consistent with the standard real-time fluorescence EXPAR. Consequently, the label-free mass-spectrometric platform could be a potential tool for miRNAs analysis in complex biological samples, and may be used for clinical diagnosis.     

A label-free differential proteomic analysis of mouse bronchoalveolar lavage fluid exposed to ultrafine carbon black

Ultrafine carbon black (ufCB) is a potential hazard to the lung. It causes changes in protein expression and it increases alveolar-capillary permeability in the lung. Label-free quantitative proteomic methods allow a sensitive and accurate analytical method for identifying and quantifying proteins in a protein mixture without chemically modifying the proteins. We used a label-free quantitative proteomic approach that combined and aligned LC-MS and LC-MS/MS spectra to analyze mouse bronchoalveolar lavage fluid (BALF) protein changes associated with exposure to ufCB. We developed a simple normalization method for quantification without spiking the internal standard. The intensities of unchanged peptides were used as normalization factors based on a statistical method to avoid the influence of peptides changed because of ufCB. LC-MS/MS spectra and then database searching were used to identify proteins. The relative abundances of the aligned peptides of identified proteins were determined using LC-MS spectra. We identified 132 proteins, of which 77 are reported for the first time. In addition, the expression of 15 inflammatory proteins and surfactant-associated proteins was regulated (i.e., 7 upregulated and 8 downregulated) compared with the controls. Several proteins not previously reported provide complementary information on the proteins present in mouse BALF, and they are potential biomarkers for the understanding of mechanisms involved in ufCB-induced lung disorders hypothesize that using the label-free quantitative proteomic approach introduced here is well suited for more rigorous, large-scale quantitative analysis of biological samples. We hypothesize that this label-free quantitative proteomic approach will be suited for a large-scale quantitative analysis of biological samples.     

Peptide and protein assays using customizable bio-affinity arrays combined with ambient ionization mass spectrometry

High-throughput identification and quantification of protein/peptide biomarkers from biofluids in a label-free manner is achieved by interfacing bio-affinity arrays (BAAs) with nano-electrospray desorption electrospray ionization mass spectrometry (nano-DESI-MS). A wide spectrum of proteins and peptides ranging from phosphopeptides to cis-diol biomolecules as well as thrombin can be rapidly extracted via arbitrarily predefined affinity interactions including coordination chemistry, covalent bonding, and biological recognition. An integrated MS platform allows continuous interrogation. Profiling and quantitation of dysregulated phosphopeptides from small-volume (∼5 μL) serum samples has been successfully demonstrated. As a front-end device adapted to any mass spectrometer, this MS platform might hold much promise in protein/peptide analysis in point-of-care (POC) diagnostics and clinical applications.

Customizable bio-affinity arrays were interfaced with ambient ionization mass spectrometry for high-throughput assays of protein/peptide biomarkers in biofluids.     

MassUntangler: a novel alignment tool for label-free liquid chromatography-mass spectrometry proteomic data

Liquid chromatography-mass spectrometry (LC-MS) has become an important analytical tool for quantitative proteomics and biomarker discovery. In the label-free differential LC-MS approach computational methods are required for an accurate alignment of peaks extrapolated from the experimental raw data accounting for retention time and m/z signals intensity, which are strongly affected by sample matrix and instrumental performance. A novel procedure "MassUntangler" for pairwise alignment has been developed, relying on a pattern-based matching algorithm integrated with filtering algorithms in a multi-step approach. The procedure has been optimized employing a two-step approach. Firstly, low-complexity LC-MS data derived from the enzymatic digestion of two standard proteins have been analyzed. Then, the algorithm's performance has been evaluated by comparing the results with other achieved using state-of-the-art alignment tools. In the second step, our algorithm has been used for the alignment of high-complexity LC-MS data consisting of peptides obtained by an Escherichia coli lysate available from a public repository previously used for the comparison of other alignment tools. MassUntangler gave excellent results in terms of precision scores (from 80% to 93%) and recall scores (from 68% to 89%), showing performances similar and even better than the previous developed tools. Considering the mass spectrometry sensitivity and accuracy, this approach allows the identification and quantification of peptides present in a biological sample at femtomole level with high confidence. The procedure's capability of aligning LC-MS data previously corrected for distortion in retention time has been studied through a hybrid approach, in which MassUntangler was interfaced with the OpenMS TOPP tool MapAligner. The hybrid aligner yielded better results, showing that an integration of different bioinformatic approaches for accurate label-free LC-MS data alignment should be used.     

Label-free quantification of differentially expressed proteins in mouse liver cancer cells with high and low metastasis rates by a SWATH acquisition method

Label-free quantification is a valuable tool for the analysis of differentially expressed proteins identified by mass spectrometry methods.Herein,we used a new strategy:data-dependent acquisition mode identification combined with label-free quantification by SWATH acquisition mode,to study the differentially expressed proteins in mouse liver cancer metastasis cells.A total of 1528 protein groups were identified,among which 1159 protein groups were quantified and 249 protein groups were observed as differentially expressed proteins(86 proteins up-regulated and 163 down-regulated).This method provides a commendable solution for the identification and quantification of differentially expressed proteins in biological samples.     

Isotope coded protein label quantification of serum proteins—Comparison with the label-free LC-MS and validation using the MRM approach

Protein quantification based upon mass spectrometry is gaining ground in diverse applications of biological and clinical relevance. The present article focuses on one of the most complex biological fluids - serum - and provides a novel ICPL based quantification protocol. The results are compared to a label-free (data independent alternate scanning) absolute quantification method. The validation is performed using MRM based protein quantification technique. Regarding the ICPL approach, serum samples used in this study were depleted of high abundant proteins, labeled with ICPL and fractionated according to their respective pI (3-5, 5-7 and 7-12). The samples were further subjected to tryptic digestion followed by treatment with the Glu-C enzyme. The peptides were analyzed on a 2D-nano-LC system using four different concentrations of salt injections (45, 75, 150 and 500 mM ammonium acetate). The LC system was connected on-line with the electrospray ion-trap mass spectrometer. For the label-free quantification the serum samples were depleted and digested with trypsin. A proteome-wide comparison was performed using highly reproducible LC and data independent alternate scanning in conjunction with a high mass accuracy orthogonal time-of-flight mass spectrometer. Selected proteins, found by both methods, were validated using the MRM approach. For this purpose non-depleted tryptically digested serum samples were analyzed by LC coupled with a triple-quadrupole MS. The relative protein quantification using ICPL and mass spectrometry allowed for the detection of approximately 200 proteins, whereas about 2/3 of those contained the ICPL label and could therefore be quantified. Label-free approach used no fractionation, less sample and was able to identify and quantify over 110 proteins. The identified proteins covered generally 3-4 orders of magnitude of protein concentration in human serum. Changes in relative abundance of eight proteins were validated using MRM. This study, for the first time, shows the ability of the relative protein quantification based upon ICPL and 2D-LC-MS/MS to quantify serum biomarkers. It provides two additional label-free approaches that could validate and bring additional value to the label-based results, offering a starting point for comprehensive proteomics studies aiming at revealing biomarkers of clinical relevance.     

NSI and NSMT: usages of MS/MS fragment ion intensity for sensitive differential proteome detection and accurate protein fold change calculation in relative label-free proteome quantification

Although widely applied in the label-free quantification of proteomics, spectral count (SC)-based abundance measurements suffer from the narrow dynamic range of attainable ratios, leading to the serious underestimation of true protein abundance fold changes, especially when studying biological samples that exhibit very large fold changes in protein expression. MS/MS fragment ion intensity, as an alternative to SC, has recently gained acceptance as the abundance feature of protein in label-free proteomic studies. Herein, we implemented two formats of MS/MS fragment ion intensity, Spectral Index (SI) and Summed MS/MS TIC (SMT), to alleviate this particular deficiency arising from SC. Both were in forms of replacing SC in the Normalized Spectral Abundance Factor (NSAF) formula, resulting in two algorithms, abbreviated as NSI and NSMT, respectively. The necessity of the normalization process was validated using a publicly available dataset. Furthermore, when applied to another well characterized benchmark dataset, both NSI and NSMT showed improved overall accuracy over NSAF for the relative quantification of proteomes. Hereinto, NSI enabled the sensitive detection of differentially expressed proteins, while NSMT ensured accurate calculation for protein abundance fold change. Therefore, the selective use of both algorithms might facilitate the screening and quantification of potential biomarkers on the proteome scale.     

Micro- and nanomechanical sensors for environmental, chemical, and biological detection

Micro- and nanoelectromechanical systems, including cantilevers and other small scale structures, have been studied for sensor applications. Accurate sensing of gaseous or aqueous environments, chemical vapors, and biomolecules have been demonstrated using a variety of these devices that undergo static deflections or shifts in resonant frequency upon analyte binding. In particular, biological detection of viruses, antigens, DNA, and other proteins is of great interest. While the majority of currently used detection schemes are reliant on biomarkers, such as fluorescent labels, time, effort, and chemical activity could be saved by developing an ultrasensitive method of label-free mass detection. Micro- and nanoscale sensors have been effectively applied as label-free detectors. In the following, we review the technologies and recent developments in the field of micro- and nanoelectromechanical sensors with particular emphasis on their application as biological sensors and recent work towards integrating these sensors in microfluidic systems.     

A comparison of relative quantification with isobaric tags on a subset of the murine hepatic proteome using electrospray ionization quadrupole time-of-flight and matrix-assisted laser desorption/ionization tandem time-of-flight

The use of isobaric tagging for relative and absolute quantification (iTRAQ) has increased dramatically over the past few years. Many factors can affect the accuracy of quantification. Some of these include the number of biological/technical replicates, sample complexity, instrumentation, method of peptide/protein identification and the statistical techniques used for data analysis. It has been observed that the low collision energies normally used in electrospray ionization quadrupole time-of-flight (ESI QTOF) can result in low iTRAQ reporter ion abundances. We used two-way analysis of variance (ANOVA) to compare the iTRAQ ratios that were generated on an ESI QTOF and a matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI TOF/TOF). It appears that iTRAQ analyses performed on an ESI QTOF without any special modifications to instrumental parameters produce essentially the same protein ratios as those obtained on a MALDI TOF/TOF.     

Detection and quantification of proteins and cells by use of elemental mass spectrometry: progress and challenges

Much progress has been made in identification of the proteins in proteomes, and quantification of these proteins has attracted much interest. In addition to popular tandem mass spectrometric methods based on soft ionization, inductively coupled plasma mass spectrometry (ICPMS), a typical example of mass spectrometry based on hard ionization, usually used for analysis of elements, has unique advantages in absolute quantification of proteins by determination of an element with a definite stoichiometry in a protein or attached to the protein. In this Trends article, we briefly describe state-of-the-art ICPMS-based methods for quantification of proteins, emphasizing protein-labeling and element-tagging strategies developed on the basis of chemically selective reactions and/or biospecific interactions. Recent progress from protein to cell quantification by use of ICPMS is also discussed, and the possibilities and challenges of ICPMS-based protein quantification for universal, selective, or targeted quantification of proteins and cells in a biological sample are also discussed critically. We believe ICPMS-based protein quantification will become ever more important in targeted quantitative proteomics and bioanalysis in the near future.

Comparative proteomics based on stable isotope labeling and affinity selection

Disease, external stimuli (such as drugs and toxins), and mutations cause changes in the rate of protein synthesis, post-translational modification, inter-compartmental transport, and degradation of proteins in living systems. Recognizing and identifying the small number of proteins involved is complicated by the complexity of biological extracts and the fact that post-translational alterations of proteins can occur at many sites in multiple ways. It is shown here that a variety of new tools and methods based on internal standard technology are now being developed to code globally all peptides in control and experimental samples for quantification. The great advantage of these stable isotope-labeling strategies is that mass spectrometers can rapidly target those proteins that have changed in concentration for further analysis. When coupled to stable isotope quantification, targeting can be further focused through chromatographic selection of peptide classes on the basis of specific structural features. Targeting structural features is particularly useful when they are unique to types of regulation or disease. Differential displays of targeted peptides show that stimulus-specific markers are relatively easy to identify and will probably be diagnostically valuable tools.     

Label-free quantitation: A new glycoproteomics approach

We demonstrate herein a method for quantifying glycosylation changes on glycoproteins. This novel method uses MS data of characterized glycopeptides to analyze glycosylation profiles, and several quality control tests were done to demonstrate that the method is reproducible, robust, applicable to different types of glycoproteins, and tolerant of instrumental variability during ionization of the analytes. This method is unique in that it is the first label-free quantitative method specifically designed for glycopeptide analysis. It can be used to monitor changes in glycosylation in a glycosylation site-specific manner on a single glycoprotein, or it can be used to quantify glycosylation in a glycoprotein mixture. During mixture analysis, the method can discriminate between changes in glycosylation of a given protein, and changes in the glycoprotein’s concentration in the mixture. This method is useful for quantitative analyses in biochemical studies of glycoproteins, where changes in glycosylation composition can be linked to functional differences; it could also be implemented in the pharmaceutical industry, where glycosylation profiles of glycoprotein-based therapeutics must be quantified. Finally, quantification of glycopeptides is an important aspect of glycopeptide-based biomarker discovery, and our quantitative approach could be a valuable asset to this field as well, provided the compositions of the glycopeptides to be quantified are identifiable using other methods.     

Quantitative mass spectrometry in proteomics: a critical review

The quantification of differences between two or more physiological states of a biological system is among the most important but also most challenging technical tasks in proteomics. In addition to the classical methods of differential protein gel or blot staining by dyes and fluorophores, mass-spectrometry-based quantification methods have gained increasing popularity over the past five years. Most of these methods employ differential stable isotope labeling to create a specific mass tag that can be recognized by a mass spectrometer and at the same time provide the basis for quantification. These mass tags can be introduced into proteins or peptides (i) metabolically, (ii) by chemical means, (iii) enzymatically, or (iv) provided by spiked synthetic peptide standards. In contrast, label-free quantification approaches aim to correlate the mass spectrometric signal of intact proteolytic peptides or the number of peptide sequencing events with the relative or absolute protein quantity directly. In this review, we critically examine the more commonly used quantitative mass spectrometry methods for their individual merits and discuss challenges in arriving at meaningful interpretations of quantitative proteomic data.     

MeCAT—new iodoacetamide reagents for metal labeling of proteins and peptides

Besides protein identification via mass spectrometric methods, protein and peptide quantification has become more and more important in order to tackle biological questions. Methods like differential gel electrophoresis or enzyme-linked immunosorbent assays have been used to assess protein concentrations, while stable isotope labeling methods are also well established in quantitative proteomics. Recently, we developed metal-coded affinity tagging (MeCAT) as an alternative for accurate and sensitive quantification of peptides and proteins. In addition to absolute quantification via inductively coupled plasma mass spectrometry, MeCAT also enables sequence analysis via electrospray ionization tandem mass spectrometry. In the current study, we developed a new labeling approach utilizing an iodoacetamide MeCAT reagent (MeCAT-IA). The MeCAT-IA approach shows distinct advantages over the previously used MeCAT with maleinimide reactivity such as higher labeling efficiency and the lack of diastereomer formation during labeling. Here, we present a careful characterization of this new method focusing on the labeling process, which yields complete tagging with an excess of reagent of 1.6 to 1, less complex chromatographic behavior, and fragmentation characteristics of the tagged peptides using the iodoacetamide MeCAT reagent.     

组蛋白翻译后修饰无标定量方法可靠性的比较

组蛋白翻译后修饰是一种表观遗传学修饰,参与调控细胞的新陈代谢等重要生理过程。蛋白质组学发展迅速,使监控组蛋白翻译后修饰的动态变化成为可能。目前主要有3种无标定量方法（谱图计数法、峰面积积分法和信号强度法）,但何种定量方法更可靠尚未见系统性的详细报道。在稳定同位素标记细胞培养技术（SILAC）基础上,对去乙酰化酶抑制剂（SAHA）调控细胞乙酰化修饰水平的定量数据进行对比,比较3种无标定量方法对组蛋白翻译后修饰进行的定量分析,利用定量结果的标准差（SD）评估定量的可靠性,最终发现基于峰面积积分法定量的结果可靠性最高。该研究对难以进行同位素标记实验的样本分析,尤其对临床样本、大样本的组蛋白修饰谱分析具有重要参考意义。     

Current trends in quantitative proteomics – an update

Proteins can provide insights into biological processes at the functional level, so they are very promising biomarker candidates. The quantification of proteins in biological samples has been routinely used for the diagnosis of diseases and monitoring the treatment. Although large‐scale protein quantification in complex samples is still a challenging task, a great amount of effort has been made to advance the technologies that enable quantitative proteomics. Seven years ago, in 2009, we wrote an article about the current trends in quantitative proteomics. In writing this current paper, we realized that, today, we have an even wider selection of potential tools for quantitative proteomics. These tools include new derivatization reagents, novel sampling formats, new types of analyzers and scanning techniques, and recently developed software to assist in assay development and data analysis. In this review article, we will discuss these innovative methods, and their current and potential applications in proteomics. Copyright © 2017 John Wiley & Sons, Ltd.     

An insight into iTRAQ: where do we stand now?

The iTRAQ (isobaric tags for relative and absolute quantification) technique is widely employed in proteomic workflows requiring relative quantification. Here, we review the iTRAQ literature; in particular, we focus on iTRAQ usage in relation to other commonly used quantitative techniques e.g. stable isotope labelling in culture (SILAC), label-free methods and selected reaction monitoring (SRM). As a result, we identify several issues arising with respect to iTRAQ. Perhaps frustratingly, iTRAQ's attractiveness has been undermined by a number of technical and analytical limitations: it may not be truly quantitative, as the changes in abundance reported will generally be underestimated. We discuss weaknesses and strengths of iTRAQ as a methodology for relative quantification in the light of this and other technical issues. We focus on technical developments targeted at iTRAQ accuracy and precision, use of 4-plex over 8-plex reagents and application of iTRAQ to post-translational modification (PTM) workflows. We also discuss iTRAQ in relation to label-free approaches, to which iTRAQ is losing ground.     

A label-free protein microfluidic array for parallel immunoassays

A label-free protein microfluidic array for immunoassays based on the combination of imaging ellipsometry and an integrated microfluidic system is presented. Proteins can be patterned homogeneously on substrate in array format by the microfluidic system simultaneously. After preparation, the protein array can be packed in the microfluidic system which is full of buffer so that proteins are not exposed to denaturing conditions. With simple microfluidic channel junction, the protein microfluidic array can be used in serial or parallel format to analyze single or multiple samples simultaneously. Imaging ellipsometry is used for the protein array reading with a label-free format. The biological and medical applications of the label-free protein microfluidic array are demonstrated by screening for antibody-antigen interactions, measuring the concentration of the protein solution and detecting five markers of hepatitis B.     

Label-free quantitative analysis for studying the interactions between nanoparticles and plasma proteins

A shotgun proteomics approach was used to compare human plasma protein binding capability with cationic liposomes, DNA–cationic lipid complexes (lipoplexes), and lipid–polycation–DNA (LPD) complexes. Nano-high-performance liquid chromatography coupled with a high-resolution LTQ Orbitrap XL mass spectrometer was used to characterize and compare their protein corona. Spectral counting and area under curve methods were used to perform label-free quantification. Substantial qualitative and quantitative differences were found among proteins bound to the three different systems investigated. Protein variety found on lipoplexes and LPD complexes was richer than that found on cationic liposomes. There were also significant differences between the amounts of protein. Such results could help in the design of gene-delivery systems, because some proteins could be more selectively bound rather than others, and their bio-distribution could be driven in vivo for more efficient and effective gene therapy.