Dynamic Range Of Proteins Research Articles

Microfluidic protein detection and quantification using droplet morphology

Sensitive, inline detection of proteins is required for post-chromatographic analyses in proteomics, cell-based assays, and drug discovery workflows. Among the common inline methods, post-column derivatization requires chemical labels, while label-free methods are either expensive (mass spectrometry) or have limited sensitivity at small length scales (UV–Vis). This paper presents a label-free detection technique based on the concept that dissolved proteins can function as surfactants and decrease the dynamic interfacial tension (IFT) of an immiscible (water–oil) interface. Existing methods for measuring IFT, such as axisymmetric drop shape analysis (ADSA), operate in batch mode and are not suitable for continuous detection. Here we show that a microfluidic flow-focusing droplet generator operating at a frequency of > 100 Hz can track IFT changes continuously, with high temporal resolution and small detection volumes. Variations in protein concentration alter the size and shape of the drops/plugs formed, and these changes can be quantified in time using a high-speed camera and in-house image processing software. Moreover, the continuously refreshing interface alleviates issues related to surface aging. Two applications are demonstrated: (1) direct injection of a single protein into a microfluidic chip. (2) post-column detection of protein mixtures separated by high performance size exclusion chromatography (SEC HPLC). Of interest, the dynamic range of protein (bovine serum albumin, BSA) was 50 -104 μg/ml without using HPLC unit. The lowest limit of detection without HPLC unit was ~ 1 μg/ml of thyroglobulin protein in a 1 nl droplet, which equates to 1 fg of total protein. When used as a detector, the aforementioned detection method offered a sensitivity of six orders of magnitude higher than conventional UV–VIS detectors.

Early detection of Ebola virus proteins in peripheral blood mononuclear cells from infected mice

BackgroundDetection of viral ribo-nucleic acid (RNA) via real-time polymerase chain reaction (RT-PCR) is the gold standard for the detection of Ebola virus (EBOV) during acute infection. However, the earliest window for viral RNA detection in blood samples is 48–72 h post-onset of symptoms. Therefore, efforts to develop additional orthogonal assays using complementary immunological and serological technologies are still needed to provide simplified methodology for field diagnostics. Furthermore, unlike RT-PCR tests, immunoassays that target viral proteins and/or early host responses are less susceptible to sequence erosion due to viral genetic drift. Although virus is shed into the bloodstream from infected cells, the wide dynamic range of proteins in blood plasma makes this a difficult sample matrix for the detection of low-abundant viral proteins. We hypothesized that the isolation of peripheral blood mononuclear cells (PBMCs), which are the first cellular targets of the Ebola virus (EBOV), may provide an enriched source of viral proteins.MethodsA mouse infection model that employs a mouse-adapted EBOV (MaEBOV) was chosen as a proof-of-principal experimental paradigm to determine if viral proteins present in PBMCs can help diagnose EBOV infection pre-symptomatically. We employed a liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) platform to provide both high sensitivity and specificity for the detection and relative quantitation of viral proteins in PBMCs collected during MaEBOV infection. Blood samples pooled from animals at the post-infection time-points were used to determine the viral load by RT-PCR and purify PBMCs.ResultsUsing quantitative LC-MS/MS, we detected two EBOV proteins (vp40 and nucleoprotein) in samples collected on Day 2 post-infection, which was also the first day of detectable viremia via RT-PCR. These results were confirmed via western blot which was performed on identical PBMC lysates from each post-infection time point.ConclusionsWhile mass spectrometry is not currently amenable to field diagnostics, these results suggest that viral protein enrichment in PBMCs in tandem with highly sensitive immunoassays platforms, could lead to the development of a rapid, high-throughput diagnostic platform for pre-symptomatic detection of EBOV infection.

Gel Filtration Chromatography Technique as Tool of Simple Study SeminalPlasma Proteins in Domestic Animals

Seminal fluid is the liquid component of sperm, providing a safe surrounding for spermatozoa. The seminal plasma has the feature common to many other body fluid, characterized by a high dynamic range of proteins, which visualizes physiological and biochemical processes of semen. As in other body fluids, it is convenient to distinguish in the seminal plasma between proteins and non-proteins. The molecular exclusion chromatographic technique presents important points to constitutional proteins preservation, in addition to the conventional phenomenon; witch exclusion hydrophobic interactions can provide a higher resolution in the chromatogram and do not provide nonspecific interactions between protein structures. The aim was the chromatography profile to simple study of proteins seminal plasma domestic animals (Stallions, Canine, and Goat), by the technique of gel filtration chromatography. The samples (seminal plasma proteins) were chromatographed in a Superose 12 HR 10/30, equilibrated with 25 mMTris-HCl (Sigma) with 0.15 M NaCl (Sigma), pH 7.4 at room temperature in a fast performance liquid chromatography (FPLC-system), using a flow rate of 0.5 mL.min-1. There was a molecular separation of significantly different molecular weights, which makes possible the logarithmic relationship between molecular weight and the elution volume. Calibration of the gel filtration column resulted in an equation y = ax + b, where the values of a and b were 5.43 and -2.175, respectively, and the corresponding errors were 0.115 and 0.256, respectively. The experimental error was less than 5% for most of the protein molecular masses. The work showed that the gel filtration chromatography technique provided an excellent analytical repeatability, and could therefore, be a valuable tool to the study preliminary of seminal plasma.

Automated sample preparation platform for mass spectrometry-based plasma proteomics and biomarker discovery.

The identification of novel biomarkers from human plasma remains a critical need in order to develop and monitor drug therapies for nearly all disease areas. The discovery of novel plasma biomarkers is, however, significantly hampered by the complexity and dynamic range of proteins within plasma, as well as the inherent variability in composition from patient to patient. In addition, it is widely accepted that most soluble plasma biomarkers for diseases such as cancer will be represented by tissue leakage products, circulating in plasma at low levels. It is therefore necessary to find approaches with the prerequisite level of sensitivity in such a complex biological matrix. Strategies for fractionating the plasma proteome have been suggested, but improvements in sensitivity are often negated by the resultant process variability. Here we describe an approach using multidimensional chromatography and on-line protein derivatization, which allows for higher sensitivity, whilst minimizing the process variability. In order to evaluate this automated process fully, we demonstrate three levels of processing and compare sensitivity, throughput and reproducibility. We demonstrate that high sensitivity analysis of the human plasma proteome is possible down to the low ng/mL or even high pg/mL level with a high degree of technical reproducibility.

CysTRAQ — A combination of iTRAQ and enrichment of cysteinyl peptides for uncovering and quantifying hidden proteomes

Shotgun proteomics is capable of characterizing differences in both protein quality and quantity, and has been applied in various biomedical applications. Unfortunately, the high complexity and dynamic range of proteins in studied samples, clinical in particular, often hinders the identification of relevant proteins. Indeed, information-rich, low abundance proteins often remain undetected, whereas repeatedly reported altered concentrations in high abundance proteins are often ambiguous and insignificant. Several techniques have therefore been developed to overcome this obstacle and provide a deeper insight into the proteome. Here we report a novel approach, which enables iTRAQ reagent quantitation of peptides fractionated based on presence of a cysteine residue (thus CysTRAQ). For the first time, we prove that iTRAQ quantitation is fully compatible with cysteinyl peptide enrichment and is not influenced by the fractionation process. Moreover, the employment of the method combined with high-resolution TripleTOF 5600 mass spectrometer for very fast MS/MS acquisition in human amniotic fluid analysis significantly increased the number of identified proteins, which were simultaneously quantified owing to the introduction of iTRAQ labeling. We herein show that CysTRAQ is a robust and straightforward method with potential application in quantitative proteomics experiments, i.e. as an alternative to the ICAT reagent approach.

Recent developments of nanoparticle-based enrichment methods for mass spectrometric analysis in proteomics

In proteome research, rapid and effective separation strategies are essential for successful protein identification due to the broad dynamic range of proteins in biological samples. Some important proteins are often expressed in ultra low abundance, thus making the pre-concentration procedure before mass spectrometric analysis prerequisite. The main purpose of enrichment is to isolate target molecules from complex mixtures to reduce sample complexity and facilitate the subsequent analyzing steps. The introduction of nanoparticles into this field has accelerated the development of enrichment methods. In this review, we mainly focus on recent developments of using different nanomaterials for pre-concentration of low-abundance peptides/ proteins, including those containing post-translational modifications, such as phosphorylation and glycosylation, prior to mass spectrometric analysis.

Proteomic analysis of the temporal expression of bovine milk proteins during coliform mastitis and label-free relative quantification

The discovery of biomarkers in milk indicative of local inflammation or disease in the bovine mammary gland has been hindered by the extreme biological complexity of milk, the dynamic range of proteins in the matrix that renders the identification of low-abundance proteins difficult, and the challenges associated with quantifying changes during disease in the abundance of proteins for which no antibody exists. The objectives of the current study were to characterize the temporal expression of milk proteins following Escherichia coli challenge and to evaluate change in relative abundance of identified proteins using a liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) label-free semiquantitative approach. Liquid chromatography–MS/MS conducted on whey from milk samples collected just before infusion with E. coli and at 12, 18, 24, 36, 48, and 60h following infection resulted in the identification of the high- to medium-abundance proteins αS1-, αS2- β-, and κ-caseins and the whey proteins serum albumin, β-lactoglobulin, and α-lactalbumin. Additionally, a select number of lower abundance markers of inflammation were also identified, including lactoferrin, transferrin, apolipoprotein AI, fibrinogen, glycosylation-dependent cell adhesion molecule-1, peptidoglycan recognition receptor protein, and cyclic dodecapeptide-1. Normalized peptide counts for each protein identified were used to evaluate temporal changes in milk proteins following infection. For comparison with relative protein abundance determined using proteomic-based methods, changes in serum albumin, lactoferrin, and transferrin in milk during disease were also measured using ELISA. Label-free, proteomic-based quantification revealed relative changes in milk proteins that corresponded to expression profiles generated by ELISA. The results indicate that label-free LC–MS/MS methods are a viable means of tracking changes in relative protein abundance in milk during disease. Despite the identification of primarily abundant milk proteins, the results indicate that, with further refinement, LC–MS/MS could be used to evaluate temporal changes in proteins related to host response for which no antibody or ELISA currently exists.

Overcoming the dynamic range problem in mass spectrometry-based shotgun proteomics

Protein profiling using mass spectrometry technology has emerged as a powerful method for analyzing large-scale protein-expression patterns in cells and tissues. However, a number of challenges are present in proteomics research, one of the greatest being the high degree of protein complexity and huge dynamic range of proteins expressed in the complex biological mixtures, which exceeds six orders of magnitude in cells and ten orders of magnitude in body fluids. Since many important signaling proteins have low expression levels, methods to detect the low-abundance proteins in a complex sample are required. This review will focus on the fundamental fractionation and mass spectrometry techniques currently used for large-scale shotgun proteomics research.

Antibody microarrays: the crucial impact of mass transport on assay kinetics and sensitivity

Although they are superficially similar to DNA microarrays, immunoassay microarrays represent a daunting technological challenge owing to the much wider diversity of proteins. Yet, as the leading edge of bioscience migrates from genomics to proteomics, the complexity and enormous dynamic range of proteins in a cell necessitate an analytic tool with exceptional specificity and sensitivity. In theory, microspot immunoassays could fulfill this need. However, antibody microarrays have had limited success to date, and have often required a highly sensitive detection system and/or sophisticated immobilization approach to be of any use for the profiling of complex specimens. There is a solid body of work on the theory of microspot reaction kinetics, yet much of the published experimental work on protein microarray development pays insufficient attention to the kinetic aspects of this interaction. This review explains that one of the main limitations for the sensitivity of current generation microspot immunoassays is the strong dependence of antibody microspot kinetics upon mass flux to the spot. This not only involves migration of analyte in solution, but also across the surface of the solid phase. Understanding of this effect will be discussed, along with several related effects and their significance to improving existing microarray designs. It is concluded that current efforts may be too focused on areas that cannot improve performance significantly, and that other critical areas of design should receive more attention. Finally, the review addresses the question of whether ambient analyte immunoassay is truly a separate category of microspot assay, with the conclusion that this may be a flawed concept.

Utilizing Human Blood Plasma for Proteomic Biomarker Discovery

Candidate proteomic biomarker discovery from human plasma holds both incredible clinical potential as well as significant challenges. The dynamic range of proteins within plasma is known to exceed 10(10), and many potential biomarkers are likely present at lower protein abundances. At present, proteomic based MS analyses provide a dynamic range typically not exceeding approximately 10(3) in a single spectrum, and approximately 10(4)-10(6) when combined with on-line separations (e.g., reversed-phase gradient liquid chromatography), and thus are generally insufficient for low level biomarker detection directly from human plasma. This limitation is providing an impetus for the development of experimental methodologies and strategies to increase the possible number of detections within this biofluid. Discussed is the diversity of available approaches currently used by our laboratory and others to utilize human plasma as a viable medium for biomarker discovery. Various separation, depletion, enrichment, and quantitative efforts as well as recent improvements in MS capabilities have resulted in measurable improvements in the detection and identification of lower abundance proteins (by approximately 10-10(2)). Despite these improvements, further advances are needed to provide a basis for discovery of candidate biomarkers at very low levels. Continued development of depletion and enrichment techniques, coupled with improved pre-MS separations (both at the protein and peptide level) holds promise in extending the dynamic range of proteomic analysis.