Serum protein profiling by solid phase extraction and mass spectrometry: A future diagnostics tool?

Serum protein profiling by MS is a promising method for early detection of disease. Important characteristics for serum protein profiling are preanalytical factors, analytical reproducibility and high throughput. Problems related to preanalytical factors can be overcome by using standardized and rigorous sample collection and sample handling protocols. The sensitivity of the MS analysis relies on the quality of the sample; consequently, the blood sample preparation step is crucial to obtain pure and concentrated samples and enrichment of the proteins and peptides of interest. This review focuses on the serum sample preparation step prior to protein profiling by MALDI MS analysis, with particular focus on various SPE methods. The application of SPE techniques with different chromatographic properties such as RP, ion exchange, or affinity binding to isolate specific subsets of molecules (subproteomes) is advantageous for increasing resolution and sensitivity in the subsequent MS analysis. In addition, several of the SPE sample preparation methods are simple and scalable and have proven easy to automate for higher reproducibility and throughput, which is important in a clinical proteomics setting.     

Multiplex proteomic approaches to sepsis research: case studies employing new technologies

Sepsis is a multifactorial disease that provides unique challenges to the critical care physician. Diagnosis is hampered by the lack of a quantitative in vitro diagnostic test, instead, it relies on a series of clinical measures. The complex nature of the disease, with involvement of several physiologic systems, suggests a need to simultaneously monitor many clinical parameters. Novel proteomic technologies now exist that enable the multiplex measurement of multiple protein analytes from the same sample. Integration of these analytical measures with patient clinical data may provide the foundation for a better understanding of disease diagnosis, disease progression and the selection of optimal therapeutic regimen. The future challenge is the translation of these multiplex approaches from investigative research to clinical diagnostics for the greatest impact on patient treatment decisions.     

High-throughput proteomics using antibody microarrays

Antibody-based microarrays are a novel technology that hold great promise in proteomics. Microarrays can be printed with thousands of recombinant antibodies carrying the desired specificities, the biologic sample (e.g., an entire proteome) and any specifically bound analytes detected. The microarray patterns that are generated can then be converted into proteomic maps, or molecular fingerprints, revealing the composition of the proteome. Using this tool, global proteome analysis and protein expression profiling will thus provide new opportunities for biomarker discovery, drug target identification and disease diagnostics, as well as providing insights into disease biology. Intense work is currently underway to develop this novel technology platform into the high-throughput proteomic tool required by the research community.     

High-throughput proteomics using antibody microarrays

Antibody-based microarrays are a novel technology that hold great promise in proteomics. Microarrays can be printed with thousands of recombinant antibodies carrying the desired specificities, the biologic sample (e.g., an entire proteome) and any specifically bound analytes detected. The microarray patterns that are generated can then be converted into proteomic maps, or molecular fingerprints, revealing the composition of the proteome. Using this tool, global proteome analysis and protein expression profiling will thus provide new opportunities for biomarker discovery, drug target identification and disease diagnostics, as well as providing insights into disease biology. Intense work is currently underway to develop this novel technology platform into the high-throughput proteomic tool required by the research community.     

Semiconductor quantum dots for in vitro diagnostics and cellular imaging

The need for companion diagnostics, point-of-care testing (POCT) and high-throughput screening in clinical diagnostics and personalized medicine has pushed the need for more biological information from a single sample at extremely low concentrations and volumes. Optical biosensors based on semiconductor quantum dots (QDs) can answer these requirements because their unique photophysical properties are ideally suited for highly sensitive multiplexed detection. Many different biological systems have been successfully scrutinized with a large variety of QDs over the past decade but their future as widely applied commercial biosensors is still open. In this review, we highlight recent in vitro diagnostic and cellular imaging applications of QDs and discuss milestones and obstacles on their way toward integration into real-life diagnostic and medical applications.     

Evaluating the effects of preanalytical variables on the stability of the human plasma proteome

High quality clinical biospecimens are vital for biomarker discovery, verification, and validation. Variations in blood processing and handling can affect protein abundances and assay reliability. Using an untargeted LC-MS approach, we systematically measured the impact of preanalytical variables on the plasma proteome. Time prior to processing was the only variable that affected the plasma protein levels. LC-MS quantification showed that preprocessing times <6 h had minimal effects on the immunodepleted plasma proteome, but by 4 days significant changes were apparent. Elevated levels of many proteins were observed, suggesting that in addition to proteolytic degradation during the preanalytical phase, changes in protein structure are also important considerations for protocols using antibody depletion. As to processing variables, a comparison of single- vs double-spun plasma showed minimal differences. After processing, the impact ?3 freeze–thaw cycles was negligible regardless of whether freshly collected samples were processed in short succession or the cycles occurred during 14–17 years of frozen storage (−80 °C). Thus, clinical workflows that necessitate modest delays in blood processing times or employ different centrifugation steps can yield valuable samples for biomarker discovery and verification studies.     

Venous and arterial endothelial proteomics: mining for markers and mechanisms of endothelial diversity

Endothelial cells (ECs) line the inside of arterial and venous blood vessels in a continuous monolayer and have the important function of responding to environmental cues to regulate vascular tone and new blood vessel formation. They also have well-defined roles in supporting tumorigenesis, and alterations in their function lead to cardiovascular disease. Consequently, ECs have been studied extensively as a cellular model of both normal and abnormal physiology. Despite their importance and the increased utility of proteomic tools in medical research, there are relatively few publications on the topic of vascular endothelial proteomics. A thorough search of the literature mined 52 publications focused exclusively on arterial and/or venous endothelial proteomics. These studies mostly relied upon examination of whole-cell lysates from cultured human umbilical vein ECs to investigate in vitro effects of various molecules, such as VEGF in the context of altering human umbilical vein EC functions related to angiogenesis. Only a few of these publications focused solely on a proteomic characterization of ECs and our analysis further revealed a lack of published studies incorporating proteomic analysis of freshly isolated ECs from tissues or in vitro conditions that mimic in vivo variables, such as oxygen tension and shear stress. It is the purpose of this article to account for the diversity of vascular EC proteomic investigations and comment on the issues that have been and should be addressed in future work.     

Multiplex proteomic approaches to sepsis research: case studies employing new technologies

Sepsis is a multifactorial disease that provides unique challenges to the critical care physician. Diagnosis is hampered by the lack of a quantitative in vitro diagnostic test, instead, it relies on a series of clinical measures. The complex nature of the disease, with involvement of several physiologic systems, suggests a need to simultaneously monitor many clinical parameters. Novel proteomic technologies now exist that enable the multiplex measurement of multiple protein analytes from the same sample. Integration of these analytical measures with patient clinical data may provide the foundation for a better understanding of disease diagnosis, disease progression and the selection of optimal therapeutic regimen. The future challenge is the translation of these multiplex approaches from investigative research to clinical diagnostics for the greatest impact on patient treatment decisions.     

Exploiting recombinant antibodies in point-of-care (POC) diagnostics: the combinatorial advantage

Antibodies are ubiquitously deployed on in vitro diagnostic (IVD) platforms for detecting a panoply of analytes indicative of environmental and food contamination, residue adulteration and both veterinary and medical diagnostics. In the clinical realm, rapid and accurate determination of disease status is paramount. The significance of immunodiagnostic performance cannot be overemphasized and in many cases reliable diagnosis informs medical intervention which can mean the difference between patient recovery and demise. Cardiovascular disease (CVD) is the single biggest cause of adult mortality in the western world and principal burden on the healthcare services. Although the troponin (Tn) family, in particular troponin I (TnI), are regarded as the gold standard for diagnosis of myocardial damage, over the last decade much research has focused on the identification of alternative cardiac biomarker molecules that can either supplant or complement TnI metrics to add value to cardiac risk stratification criteria. In particular, markers that appear earlier than TnI in the pathophyisiology of cardiac disease are highly sought after. The subject?of this addendum represents part of a broader challenge to deliver novel rapid point-of-care (POC) diagnostics to provide a chip-based multi-plexed platform for more comprehensive profiling of cardiac status with additive diagnostic and prognostic value.?Specifically, it outlines proof-of-concept direct myeloperoxidase (MPO) detection, demonstrates the benefits of using recombinant antibodies in POC diagnostics and describes optimized strategies for generation of superior candidate antibody panels.     

Global stability of plasma proteomes for mass spectrometry-based analyses

Peptide-based mass spectrometry approaches, such as multiple reaction monitoring, provide a powerful means to measure candidate protein biomarkers in plasma. A potential confounding problem is the effect of preanalytical variables, which may affect the integrity of proteins and peptides. Although some blood proteins undergo rapid physiological proteolysis ex vivo, the stability of most plasma proteins to preanalytical variables remains largely unexplored. We applied liquid chromatography-tandem mass spectrometry shotgun proteomics and multiple reaction monitoring analyses to characterize the stability of proteins at the peptide level in plasma. We systematically evaluated the effects of delay in plasma preparation at different temperatures, multiple freeze-thaw cycles and erythocyte hemolysis on peptide and protein inventories in prospectively collected human plasma. Time course studies indicated few significant changes in peptide and protein identifications, semitryptic peptides and methionine-oxidized peptides in plasma from blood collected in EDTA plasma tubes and stored for up to a week at 4 °C or room temperature prior to plasma isolation. Similarly, few significant changes were observed in similar analyses of plasma subjected to up to 25 freeze-thaw cycles. Hemolyzed samples produced no significant differences beyond the presence of hemoglobin proteins. Finally, paired comparisons of plasma and serum samples prepared from the same patients also yielded few significant differences, except for the depletion of fibrinogen in serum. Blood proteins thus are broadly stable to preanalytical variables when analyzed at the peptide level. Collection protocols to generate plasma for multiple reaction monitoring-based analyses may have different requirements than for other analyses directed at intact proteins.     

Effects of blood sample anticoagulants on lateral flow assays using luminescent photon-upconverting and Eu(III) nanoparticle reporters

Many quantitative and semiquantitative lateral flow (LF) assays have been introduced for clinical analytes such as biomarkers for cancer or acute myocardial infarction (AMI). Various detection technologies involving quantitative analyzing devices have been reported to have sufficient analytical sensitivity and quantification capability for clinical point-of-care tests. Fluorescence-based detection technologies such as quantum dots, Eu(III) nanoparticles, and photon-upconverting nanoparticles (UCNPs) have been introduced as promising solutions for point-of-care devices because of their high detectability by optical sensors. Lateral flow assays can be used for various sample types, e.g., urine, saliva, cerebrospinal fluid, and blood. This study focuses on the properties of serum and plasma because of their relevance in cancer and AMI diagnostics. The limit of detection was compared in LF assays having Eu(III) nanoparticles or UCNPs as reporters and the antibody configurations for two different analytes (prostate-specific antigen and cardiac troponin I (cTnI)). The results indicate a significant effect of anticoagulants in venipuncture tubes. The samples in K₃EDTA tubes resulted in significant interference by decreased reporter particle mobility, and thus the limit of detection was up to eightfold less sensitive compared to serum samples. Despite the matrix interference in the cTnI assay with UCNP reporters, limits of detection of 41 ng/L with serum and 66 ng/L with the Li-heparin sample were obtained.     

A proteomics performance standard to support measurement quality in proteomics

The emergence of MS-based proteomic platforms as a prominent technology utilized in biochemical and biomedical research has increased the need for high-quality MS measurements. To address this need, National Institute of Standards and Technology (NIST) reference material (RM) 8323 yeast protein extract is introduced as a proteomics quality control material for benchmarking the preanalytical and analytical performance of proteomics-based experimental workflows. RM 8323 yeast protein extract is based upon the well-characterized eukaryote Saccharomyces cerevisiae and can be utilized in the design and optimization of proteomics-based methodologies from sample preparation to data analysis. To demonstrate its utility as a proteomics quality control material, we coupled LC-MS/MS measurements of RM 8323 with the NIST MS Quality Control (MSQC) performance metrics to quantitatively assess the LC-MS/MS instrumentation parameters that influence measurement accuracy, repeatability, and reproducibility. Due to the complexity of the yeast proteome, we also demonstrate how NIST RM 8323, along with the NIST MSQC performance metrics, can be used in the evaluation and optimization of proteomics-based sample preparation methods.     

Proteomics: from basic research to diagnostic application. A review of requirements & needs

For several years proteomics research has been expected to lead to the finding of new markers that will translate into clinical tests applicable to samples such as serum, plasma and urine: so-called in vitro diagnostics (IVDs). Attempts to implement technologies applied in proteomics, in particular protein arrays and surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF MS), as IVD instruments have initiated constructive discussions on opportunities and challenges inherent in such a translation process also with respect to the use of multi-marker profiling approaches and pattern signatures in IVD. Taking into account the role that IVD plays in health care, we describe IVD requirements and needs. Subject to stringent costs versus benefit analyses, IVD has to provide reliable information about a person's condition, prognosis or risk to suffer a disease, thus supporting decisions on treatment or prevention. It is mandatory to fulfill requirements in routine IVD, including disease prevention, diagnosis, prognosis, and treatment monitoring or follow up among others. To fulfill IVD requirements, it is essential to (1) provide diagnostic tests that allow for definite and reliable diagnosis tied to a decision on interventions (prevention, treatment, or nontreatment), (2) meet stringent performance characteristics for each analyte (in particular test accuracy, including both precision of the measurement and trueness of the measurement), and (3) provide adequate diagnostic accuracy, i.e., diagnostic sensitivity and diagnostic specificity, determined by the desired positive and negative predictive values which depend on disease frequency. The fulfillment of essential IVD requirements is mandatory in the regulated environment of modern diagnostics. Addressing IVD needs at an early stage can support a timely and effective transition of findings and developments into routine diagnosis. IVD needs reflect features that are useful in clinical practice. This helps to generate acceptance and assists the implementation process. On the basis of IVD requirements and needs, we outline potential implications for clinical proteomics focused on applied research activities.     

Urinary proteomics: towards biomarker discovery, diagnostics and prognostics

During recent years, the proteomics field has moved onward to clinical applications, particularly for biomarker discovery, diagnostics and prognostics of human diseases. The urine is one of the ideal clinical samples for such applications because it is readily available in almost all patients, and its collection is very simple and non-invasive. Urinary proteomics thus becomes one of the most interesting subdisciplines in the clinical proteomics area. This article highlights and updates recent progress in the urinary proteomics field for clinical applications.     

Proteomics under pressure: development of essential sample preparation techniques in proteomics using ultrahigh hydrostatic pressure

The minimization of preanalytical variables in sample preparation is imperative for successful discovery-driven and translational research involving large-scale biomolecular profiling. Here, we demonstrate a novel technique using high hydrostatic pressure in addition to several chaotropes and solvents to maximize efficiency of both cell lysis and enzymatic digestion while minimizing the time, manual involvement in sample processing, and preanalytical variability introduced prior to mass spectrometry-based proteomic analysis. The digestion techniques were evaluated and optimized for in-solution, in-gel, and on-membrane applications using protein standards and cell lysates. The lysis techniques were evaluated using human HepG2 cells. Our results demonstrate that the use of elevated pressure and organic solvents can achieve superior protein recovery of organelle-, complex-, and especially membrane-associated proteins, meanwhile obtaining more than a 20-fold increase in throughput with improved reproducibility. This study introduces the concept of ultrahigh-performance sample preparation platforms for targeted characterization of proteome subsets in biological systems.     

Proteomics and its applications for biomarker discovery in human saliva

Human saliva is a biological fluid with enormous diagnostic potential. Because saliva can be non-invasively collected, it provides an attractive alternative for blood, serum or plasma. It has been postulated that the blood concentrations of many components are reflected in saliva. Saliva harbors a wide array of proteins, which can be informative for the detection of diseases. Profiling the proteins in saliva over the course of disease progression could reveal potential biomarkers indicative of different stages of diseases, which may be useful in medical diagnostics. With advanced instrumentation and developed refined analytical techniques, proteomics is widely envisioned as a useful and powerful approach for salivary proteomic biomarker discovery. As proteomic technologies continue to mature, salivary proteomics have great potential for biomarker research and clinical applications. The progress and current status of salivary proteomics and its application in the biomarker discovery of oral and systematic diseases will be reviewed. The scientific and clinical challenges underlying this approach will also be discussed.     

Proteomics and its applications for biomarker discovery in human saliva

Human saliva is a biological fluid with enormous diagnostic potential. Because saliva can be non-invasively collected, it provides an attractive alternative for blood, serum or plasma. It has been postulated that the blood concentrations of many components are reflected in saliva. Saliva harbors a wide array of proteins, which can be informative for the detection of diseases. Profiling the proteins in saliva over the course of disease progression could reveal potential biomarkers indicative of different stages of diseases, which may be useful in medical diagnostics. With advanced instrumentation and developed refined analytical techniques, proteomics is widely envisioned as a useful and powerful approach for salivary proteomic biomarker discovery. As proteomic technologies continue to mature, salivary proteomics have great potential for biomarker research and clinical applications. The progress and current status of salivary proteomics and its application in the biomarker discovery of oral and systematic diseases will be reviewed. The scientific and clinical challenges underlying this approach will also be discussed.