Rarity gives a charm: evaluation of trace proteins in plasma and serum

Since plasma potentially contacts every cell as it circulates through the body, it may carry clues both to diagnosis and treatment of disease. It is commonly expected that the growing ability to detect and characterize trace proteins will result in discovery of novel therapeutics and biomarkers; however, the familiar, super-abundant plasma proteins remain a fundamental stumbling block. Furthermore, robust validation of proteomic data is a sometimes overlooked but always necessary component for the eventual development of clinical reagents. This review surveys some of the uses of typical and atypical low-abundance proteins, current analytical methods, existing impediments to discovery, and some innovations that are overcoming the challenges to evaluation of trace proteins in plasma and serum.     

Serum/Plasma depletion with chicken immunoglobulin Y antibodies for proteomic analysis from multiple Mammalian species.

Plasma from different species is the most accessible and valuable source for biomarker discovery in clinical and animal samples. However, due to the high abundance of some proteins such as albumin and immunoglobulins, low-abundant proteins are often undetectable in proteomic analysis of plasma. We have established a plasma depletion scheme using chicken antibodies against various abundant proteins. This immunoaffinity purification procedure is able to deplete albumin across multiple species. The high binding capacity and specificity of the chicken antibody enables the efficient capture of its ligand from microliter volumes of plasma sample. The resulting two-dimensional gel analyses of the depleted and captured samples show significant enhancement of the low-abundant proteins and specific capture of the abundant ligand. By utilizing this sample preparation scheme, it is now possible to analyze the plasma proteome from multiple species in a potentially rapid and large-scale capacity for biomarker discovery, drug target discovery, and toxicology studies.     

A systematic analysis of eluted fraction of plasma post immunoaffinity depletion: implications in biomarker discovery

Plasma is the most easily accessible source for biomarker discovery in clinical proteomics. However, identifying potential biomarkers from plasma is a challenge given the large dynamic range of proteins. The potential biomarkers in plasma are generally present at very low abundance levels and hence identification of these low abundance proteins necessitates the depletion of highly abundant proteins. Sample pre-fractionation using immuno-depletion of high abundance proteins using multi-affinity removal system (MARS) has been a popular method to deplete multiple high abundance proteins. However, depletion of these abundant proteins can result in concomitant removal of low abundant proteins. Although there are some reports suggesting the removal of non-targeted proteins, the predominant view is that number of such proteins is small. In this study, we identified proteins that are removed along with the targeted high abundant proteins. Three plasma samples were depleted using each of the three MARS (Hu-6, Hu-14 and Proteoprep 20) cartridges. The affinity bound fractions were subjected to gelC-MS using an LTQ-Orbitrap instrument. Using four database search algorithms including MassWiz (developed in house), we selected the peptides identified at <1% FDR. Peptides identified by at least two algorithms were selected for protein identification. After this rigorous bioinformatics analysis, we identified 101 proteins with high confidence. Thus, we believe that for biomarker discovery and proper quantitation of proteins, it might be better to study both bound and depleted fractions from any MARS depleted plasma sample.     

Biomarker discovery from the plasma proteome using multidimensional fractionation proteomics

Because biomarkers are typically low in abundance, the crucial step of biomarker discovery is to efficiently separate clinically relevant sets of proteins that might define disease stages and/or predict disease development. It is anticipated that a multi-dimensional fractionation system (MDFS) will provide an efficient means of separating low abundance proteins from plasma proteins, resulting in the extension of the detection limit. However, when using an MDFS to analyze the plasma proteome it is important to consider how sample processing, yield, resolution and throughput potential may influence the detection limit. This review evaluates the recent advances in MDFS research with respect to '4RS criterion' (4R: resolution, reproducibility, recovery, and robustness; 4S: simplicity, speed, selectivity and sensitivity) and discusses perspectives for future plasma-derived biomarker discovery.     

Hexadecanedionic acid-sepharose 4B: A new tool for preparation of albumin-depleted plasma

Serum and plasma are the major sources of human material for clinical molecular diagnostics and drug discovery. However, due to the high abundance of some proteins, of which serum albumin (SA) is most prominent, lower-abundance proteins often remain undetectable in proteomic analysis of these body fluids. We have used hexadecanedionic acid (HDA) immobilized to Sepharose 4B to develop an affinity resin that is effective in the removal of SA from plasma. Two-dimensional gel analysis of the SA-depleted samples shows a significant enhancement of the low-abundance proteins and highly specific capture of serum albumin. The HDA resin shows better performance in terms of specificity than dye-based resins.     

A new method for isolation of interstitial fluid from human solid tumors applied to proteomic analysis of ovarian carcinoma tissue

Major efforts have been invested in the identification of cancer biomarkers in plasma, but the extraordinary dynamic range in protein composition, and the dilution of disease specific proteins make discovery in plasma challenging. Focus is shifting towards using proximal fluids for biomarker discovery, but methods to verify the isolated sample's origin are missing. We therefore aimed to develop a technique to search for potential candidate proteins in the proximal proteome, i.e. in the tumor interstitial fluid, since the biomarkers are likely to be excreted or derive from the tumor microenvironment. Since tumor interstitial fluid is not readily accessible, we applied a centrifugation method developed in experimental animals and asked whether interstitial fluid from human tissue could be isolated, using ovarian carcinoma as a model. Exposure of extirpated tissue to 106 g enabled tumor fluid isolation. The fluid was verified as interstitial by an isolated fluid:plasma ratio not significantly different from 1.0 for both creatinine and Na(+), two substances predominantly present in interstitial fluid. The isolated fluid had a colloid osmotic pressure 79% of that in plasma, suggesting that there was some sieving of proteins at the capillary wall. Using a proteomic approach we detected 769 proteins in the isolated interstitial fluid, sixfold higher than in patient plasma. We conclude that the isolated fluid represents undiluted interstitial fluid and thus a subproteome with high concentration of locally secreted proteins that may be detected in plasma for diagnostic, therapeutic and prognostic monitoring by targeted methods.     

Optimizing Size Exclusion Chromatography for Extracellular Vesicle Enrichment and Proteomic Analysis from Clinically Relevant Samples

The field of extracellular vesicle (EV) research has rapidly expanded in recent years, with particular interest in their potential as circulating biomarkers. Proteomic analysis of EVs from clinical samples is complicated by the low abundance of EV proteins relative to highly abundant circulating proteins such as albumin and apolipoproteins. To overcome this, size exclusion chromatography (SEC) has been proposed as a method to enrich EVs whilst depleting protein contaminants; however, the optimal SEC parameters for EV proteomics have not been thoroughly investigated. Here, quantitative evaluation and optimization of SEC are reported for separating EVs from contaminating proteins. Using a synthetic model system followed by cell line‐derived EVs, it is found that a 10 mL Sepharose 4B column in PBS produces optimal resolution of EVs from background protein. By spiking‐in cancer cell‐derived EVs to healthy plasma, it is shown that some cancer EV‐associated proteins are detectable by nano‐LC‐MS/MS when as little as 1% of the total plasma EV number are derived from a cancer cell line. These results suggest that an optimized SEC and nanoLC‐MS/MS workflow may be sufficiently sensitive for disease EV protein biomarker discovery from patient‐derived clinical samples.     

Development of quantitative plasma N-glycoproteomics using label-free 2-D LC-MALDI MS and its applicability for biomarker discovery in hepatocellular carcinoma

There has been rapid progress in the development of clinical proteomic methodologies with improvements in mass spectrometric technologies and bioinformatics, leading to many new methodologies for biomarker discovery from human plasma. However, it is not easy to find new biomarkers because of the wide dynamic range of plasma proteins and the need for their quantification. Here, we report a new methodology for relative quantitative proteomic analysis combining large-scale glycoproteomics with label-free 2-D LC-MALDI MS. In this method, enrichment of glycopeptides using hydrazide resin enables focusing on plasma proteins with lower abundance corresponding to the tissue leakage region. On quantitative analysis, signal intensities by 2-D LC-MALDI MS were normalized using a peptide internal control, and the values linked to LC data were treated with DeView? software. Our proteomic method revealed that the quantitative dynamic ranged from 102 to 10? pg/mL of plasma proteins with good reproducibility, and the limit of detection was of the order of a few ng/mL of proteins in biological samples. To evaluate the applicability of our method for biomarker discovery, we performed a feasibility study using plasma samples from patients with hepatocellular carcinoma, and identified biomarker candidates, including ceruloplasmin, alpha-1 antichymotrypsin, and multimerin-1.     

Intracellular trafficking of TRP channels

Thirteen years ago, it was suggested that exocytotic insertion of store-operated channels into the plasma membrane lead to increased Ca(2+) entry in non-excitable cells upon G protein-coupled or tyrosine kinase receptor stimulation. Since the discovery of the TRP channel superfamily and their involvement in receptor-induced Ca(2+) entry, many studies have shown that different members of the TRP superfamily translocate into the plasma membrane upon stimulation. While the exact molecular mechanism by which TRP channels insert into the plasma membrane is unknown, TRP-binding proteins have been shown to directly regulate this trafficking. This review summarizes recent advances related to the mechanism of TRP channel trafficking, focusing on the role of TRP-binding proteins.     

Peptides and proteins in plasma and cerebrospinal fluid as biomarkers for the prediction, diagnosis, and monitoring of therapeutic efficacy of Alzheimer's disease

Alzheimer's disease (AD) affects millions of persons worldwide. Earlier detection and/or diagnosis of AD would permit earlier intervention, which conceivably could delay progression of this dementing disorder. In order to accomplish this goal, reliable and specific biomarkers are needed. Biomarkers are multidimensional and have the potential to aid in various facets of AD such as diagnostic prediction, assessment of disease stage, discrimination from normally cognitive controls as well as other forms of dementia, and therapeutic efficacy of AD drugs. To date, biomarker research has focused on plasma and cerebrospinal fluid (CSF), two bodily fluids believed to contain the richest source of biomarkers for AD. CSF is the fluid surrounding the central nervous system (CNS), and is the most indicative obtainable fluid of brain pathology. Blood plasma contains proteins that affect brain processes from the periphery, as well as proteins/peptides exported from the brain; this fluid would be ideal for biomarker discovery due to the ease and non-invasive process of sample collection. However, it seems reasonable that biomarker discovery will result in combinations of CSF, plasma, and other fluids such as urine, to serve the aforementioned purposes. This review focuses on proteins and peptides identified from CSF, plasma, and urine that may serve as biomarkers in AD.     

A sensitive urea-silver stain method for detecting trace quantities of separated proteins in polyacrylamide gels

An ultrasensitive method using a urea-silver staining procedure to detect trace quantities of proteins in polyacrylamide gels (PAGE) is described. This technique is sensitive enough to detect picogram quantities of proteins resolved on sodium dodecyl sulfate-polyacrylamide gels. The major advantages of our method are that it provides a clear background and it is more sensitive than other techniques allowing it to substitute for radioisotopic techniques in some cases.     

Reducing protein concentration range of biological samples using solid-phase ligand libraries

The discovery of specific polypeptides of diagnostic relevance from a biological liquid is complicated by the overall vast number and the large concentration range of all polypeptides/proteins in the sample. Depletion or fractionation methodologies have been used for selectively removing abundant proteins; however, they failed to significantly enrich trace proteins. Here we expand upon a new method that allows the reduction of the protein concentration range within a complex mixture, like neat serum, through the simultaneous dilution of high abundance proteins and the concentration of low abundance ones in a single, simple step. This methodology utilizes solid-phase ligand libraries of large diversity. With a controlled sample-to-ligand ratio it is possible to modulate the relative concentration of proteins such that a large number of peptides or proteins that are normally not detectable by classical analytical methods become, easily detectable. Application of this method for reducing the dynamic range of unfractionated serum is specifically described along with treatment of other biological extracts. Analytical surface enhanced laser desorption/ionization mass spectrometry (SELDI-MS) technology and mono- and two-dimensional electrophoresis (1-DE and 2-DE) demonstrate the increase in the number of proteins detected. Examples linking this approach with additional fractionation methods demonstrate a further increase in the number of detectable species using either the so-called "top down" or "bottom up" approaches for proteomics analysis. By enabling the detection of a greater proportion of polypeptides/proteins within a sample, this method may contribute significantly towards the discovery of new biomarkers of diagnostic relevance.     

Middle Jurassic tetrapod burrows preserved in association with the large sauropod Omeisaurus jiaoi from the Sichuan Basin,China

Here we report a Jurassic tetrapod burrow preserved in association with the partial skeleton of a large sauropod specimen of Omeisaurus jiaoi from Zigong, Sichuan Province, China. The ichnofossil can be divided into two parts, which may indicate two individual trace makers and some social behavior, although the possibility that they are two portions of one trace by a single trace maker cannot be ruled out. The burrow trace was examined via petrographic thin sections and carbonate analysis. Considering the spatial relationship of the burrows and the skeleton, it is likely that decomposition of the sauropod carcass preceded the formation of the burrows. It is possible that the process of decomposition improved the humus level of the soil, which would have attracted more soil-dwelling invertebrates and, by consequence, tetrapod predators thereof. The discovery of ZDM5051 has increased our understanding of global ichnofossil diversity.     

Multiplexed LC‐MS/MS analysis of horse plasma proteins to study doping in sport

The development of protein biomarkers for the indirect detection of doping in horse is a potential solution to doping threats such as gene and protein doping. A method for biomarker candidate discovery in horse plasma is presented using targeted analysis of proteotypic peptides from horse proteins. These peptides were first identified in a novel list of the abundant proteins in horse plasma. To monitor these peptides, an LC‐MS/MS method using multiple reaction monitoring was developed to study the quantity of 49 proteins in horse plasma in a single run. The method was optimised and validated, and then applied to a population of race‐horses to study protein variance within a population. The method was finally applied to longitudinal time courses of horse plasma collected after administration of an anabolic steroid to demonstrate utility for hypothesis‐driven discovery of doping biomarker candidates.     

2D-electrophoresis and the urine proteome map: Where do we stand?

The discovery of urinary biomarkers is a main topic in clinical medicine. The development of proteomics has rapidly changed the knowledge on urine protein composition and probably will modify it again. Two-dimensional electrophoresis (2D-PAGE) coupled with mass spectrometry has represented for years the technique of choice for the analysis of urine proteins and it is time to draw some conclusions.This review will focus on major methodological aspects related to urine sample collection, storage and analysis by 2D-PAGE and attempt to define an advanced normal urine protein map.Overall, 1118 spots were reproducibly found in normal urine samples but only 275 were characterized as isoforms of 82 proteins. One-hundred height spots belonging to 30 proteins were also detected in plasma and corresponded to typical plasma components. The identity of most of the proteins found in normal urine by 2D-PAGE remains to be determined, the majority being low-molecular weight proteins (< 30 kDa). Equalization procedures would also enhance sensitivity of the analysis and allow low abundance proteins to be characterized.Therefore, we are still on the way to define the normal urine composition. Technology advancements in concentrating procedure will improve sensitivity and give the possibility to purify proteins for mass spectrometry.     

Integrated pipeline for mass spectrometry-based discovery and confirmation of biomarkers demonstrated in a mouse model of breast cancer

Despite their potential to impact diagnosis and treatment of cancer, few protein biomarkers are in clinical use. Biomarker discovery is plagued with difficulties ranging from technological (inability to globally interrogate proteomes) to biological (genetic and environmental differences among patients and their tumors). We urgently need paradigms for biomarker discovery. To minimize biological variation and facilitate testing of proteomic approaches, we employed a mouse model of breast cancer. Specifically, we performed LC-MS/MS of tumor and normal mammary tissue from a conditional HER2/Neu-driven mouse model of breast cancer, identifying 6758 peptides representing >700 proteins. We developed a novel statistical approach (SASPECT) for prioritizing proteins differentially represented in LC-MS/MS datasets and identified proteins over- or under-represented in tumors. Using a combination of antibody-based approaches and multiple reaction monitoring-mass spectrometry (MRM-MS), we confirmed the overproduction of multiple proteins at the tissue level, identified fibulin-2 as a plasma biomarker, and extensively characterized osteopontin as a plasma biomarker capable of early disease detection in the mouse. Our results show that a staged pipeline employing shotgun-based comparative proteomics for biomarker discovery and multiple reaction monitoring for confirmation of biomarker candidates is capable of finding novel tissue and plasma biomarkers in a mouse model of breast cancer. Furthermore, the approach can be extended to find biomarkers relevant to human disease.     

The human serum proteome: display of nearly 3700 chromatographically separated protein spots on two-dimensional electrophoresis gels and identification of 325 distinct proteins

Plasma, the soluble component of the human blood, is believed to harbor thousands of distinct proteins, which originate from a variety of cells and tissues through either active secretion or leakage from blood cells or tissues. The dynamic range of plasma protein concentrations comprises at least nine orders of magnitude. Proteins involved in coagulation, immune defense, small molecule transport, and protease inhibition, many of them present in high abundance in this body fluid, have been functionally characterized and associated with disease processes. For example, protein sequence mutations in coagulation factors cause various serious disease states. Diagnosing and monitoring such diseases in blood plasma of affected individuals has typically been conducted by use of enzyme-linked immunosorbent assays, which using a specific antibody quantitatively measure only the affected protein in the tested plasma samples. The discovery of protein biomarkers in plasma for diseases with no known correlations to genetic mutations is challenging. It requires a highly parallel display and quantitation strategy for proteins. We fractionated blood serum proteins prior to display on two-dimensional electrophoresis (2-DE) gels using immunoaffinity chromatography to remove the most abundant serum proteins, followed by sequential anion-exchange and size-exclusion chromatography. Serum proteins from 74 fractions were displayed on 2-DE gels. This approach succeeded in resolving approximately 3700 distinct protein spots, many of them post-translationally modified variants of plasma proteins. About 1800 distinct serum protein spots were identified by mass spectrometry. They collapsed into 325 distinct proteins, after sequence homology and similarity searches were carried out to eliminate redundant protein annotations. Although a relatively insensitive dye, Coomassie Brilliant Blue G-250, was used to visualize protein spots, several proteins known to be present in serum in < 10 ng/mL concentrations were identified such as interleukin-6, cathepsins, and peptide hormones. Considering that our strategy allows highly parallel protein quantitation on 2-DE gels, it holds promise to accelerate the discovery of novel serum protein biomarkers.     

High dynamic range characterization of the trauma patient plasma proteome

Although human plasma represents an attractive sample for disease biomarker discovery, the extreme complexity and large dynamic range in protein concentrations present significant challenges for characterization, candidate biomarker discovery, and validation. Herein we describe a strategy that combines immunoaffinity subtraction and subsequent chemical fractionation based on cysteinyl peptide and N-glycopeptide captures with two-dimensional LC-MS/MS to increase the dynamic range of analysis for plasma. Application of this "divide-and-conquer" strategy to trauma patient plasma significantly improved the overall dynamic range of detection and resulted in confident identification of 22,267 unique peptides from four different peptide populations (cysteinyl peptides, non-cysteinyl peptides, N-glycopeptides, and non-glycopeptides) that covered 3,654 different proteins with 1,494 proteins identified by multiple peptides. Numerous low abundance proteins were identified, exemplified by 78 "classic" cytokines and cytokine receptors and by 136 human cell differentiation molecules. Additionally a total of 2,910 different N-glycopeptides that correspond to 662 N-glycoproteins and 1,553 N-glycosylation sites were identified. A panel of the proteins identified in this study is known to be involved in inflammation and immune responses. This study established an extensive reference protein database for trauma patients that provides a foundation for future high throughput quantitative plasma proteomic studies designed to elucidate the mechanisms that underlie systemic inflammatory responses.     

Identification of Glycoproteins from Mouse Skin Tumors and Plasma

Plasma has been the focus of testing different proteomic technologies for the identification of biomarkers due to its ready accessibility. However, it is not clear if direct proteomic analysis of plasma can be used to discover new marker proteins from tumor that are associated with tumor progression. Here, we reported that such proteins can be detected in plasma in a chemical induced skin cancer mouse model. We analyzed glycoproteins from both benign papillomas and malignant carcinomas from mice using our recently developed platform, solid-phase extraction of glycopeptides (SPEG) and mass spectrometry, and identified 463 unique N-linked glycosites from 318 unique glycoproteins. These include most known extracellular proteins that have been reported to play roles in skin cancer development such as thrombospondin, cathepsins, epidermal growth factor receptor, cell adhesion molecules, cadherins, integrins, tuberin, fibulin, TGFβ receptor, etc. We further investigated whether these tumor proteins could be detected in plasma from tumor bearing mice using isotope labeling and 2D-LC-MALDI-MS/MS. Two tumor glycoproteins, Tenascin-C and Arylsulfatase B, were identified and quantified successfully in plasma from tumor bearing mice. This result indicates that analysis of tumor associated proteins in tumors and plasma by method using glycopeptide capture, isotopic labeling, and mass spectrometry can be used as a discovery tool to identify candidate tumor proteins that may be detected in plasma.