Abundant Plasma Protein Depletion Research Articles

Depletion of abundant plasma proteins for extracellular vesicle proteome characterization: benefits and pitfalls

Blood extracellular vesicles (EVs) play essential roles in cell–cell communication and their molecular cargo is a promising source of disease biomarkers. However, proteomic characterization of plasma-derived EVs is challenged by the presence of highly abundant plasma proteins, which limits the detection of less abundant proteins, and by the low number of EVs in biological fluids. The aim of this study was to investigate if the removal of abundant plasma proteins prior to EV isolation could improve plasma-derived EV characterization by LC–MS/MS and expand the proteome coverage. Plasma depletion was performed using a single-use spin column and EVs were isolated from only 100 µL of non-depleted and depleted plasma by size exclusion chromatography. Afterwards, EVs were characterized by nanoparticle tracking analysis and mass spectrometry–based proteomics using a data-independent acquisition approach. Depleted plasma-derived EVs had higher particle concentrations and particle-to-protein ratios. Depletion did increase the protein coverage with a higher number of identifications in EVs from depleted plasma (474 proteins) than from non-depleted (386 proteins). However, EVs derived from non-depleted plasma carried a slightly higher number of common EV markers. Overall, our findings suggest that plasma depletion prior to EV isolation by size exclusion chromatography provides higher yield and protein coverage, but slightly lower identification of EV markers. This study also showed the possibility to characterize the proteome of EVs derived from small plasma volumes, encouraging the clinical feasibility of the discovery of EV biomarkers.

Elevated plasma α1-antichymotrypsin is a biomarker candidate for malaria patients

Advancements in the field of proteomics have provided opportunities to develop diagnostic and therapeutic strategies against various diseases. About half of the world's population remains at risk of malaria. Caused by protozoan parasites of the genus Plasmodium, malaria is one of the oldest and largest risk factors responsible for the global burden of infectious diseases with an estimated 3.2 billion persons at risk of infection. For epidemiological surveillance and appropriate treatment of individuals infected with Plasmodium spp., timely detection is critical. In this study, we used combinations of depletion of abundant plasma proteins, 2-dimensional gel electrophoresis (2-DE), image analysis, LC-MS/MS and western blot analysis on the plasma of healthy donors (100 individuals) and vivax and falciparum malaria patients (100 vivax malaria patients and 8 falciparum malaria patients). These analyses revealed that α1-antichymotrypsin (AACT) protein levels were elevated in vivax malaria patient plasma samples (mean fold-change ± standard error: 2.83 ± 0.11, based on band intensities), but not in plasma from patients with other mosquito-borne infectious diseases. The results of AACT immunoblot analyses showed that AACT protein was significantly elevated in vivax and falciparum malaria patient plasma samples (≥ 2-fold) compared to healthy control donor plasma samples, which has not been previously reported. [BMB Reports 2022; 55(11): 571-576].

Proteomic discovery in sickle cell disease: Elevated neurogranin levels in children with sickle cell disease.

Purpose:Sickle cell disease (SCD) is an inherited hemoglobinopathy that causes stroke and silent cerebral infarct (SCI). Our aim was to identify markers of brain injury in SCD.Experimental Design:Plasma proteomes were analyzed using a sequential separation approach of hemoglobin (Hb) and top abundant plasma protein depletion, followed by reverse phase separation of intact proteins, trypsin digestion, and tandem mass spectrometry. We compared plasma proteomes of children with SCD with and without SCI in the Silent Cerebral Infarct Multi-Center Clinical Trial (SIT Trial) to age-matched, healthy non-SCD controls.Results:From the SCD group, 1172 proteins were identified. Twenty-five percent (289/1172) were solely in the SCI group. Twenty-five proteins with enriched expression in the human brain were identified in the SCD group. Neurogranin (NRGN) was the most abundant brain-enriched protein in plasma of children with SCD. Using a NRGN sandwich immunoassay and SIT Trial samples, median NRGN levels were higher at study entry in children with SCD (0.28 ng/mL, N = 100) compared to control participants (0.12 ng/mL, N = 25, p < 0.0004).Conclusions and Clinical Relevance:NRGN levels are elevated in children with SCD. NRGN and other brain-enriched plasma proteins identified in plasma of children with SCD may provide biochemical evidence of neurological injury.

Proteomic Discovery: Elevated Neurogranin Levels in Children with Sickle Cell Disease

Sickle cell disease (SCD) is an inherited hemoglobinopathy that frequently causes neurological complications, such as stroke, silent cerebral infarct (SCI) and other forms of brain injury, including loss of cognition. SCI is defined as any ischemic lesion visible on T2-weighted magnetic resonance imaging (MRI) that is not associated with a focal neurologic deficit in the same vascular distribution, and is the most commonly recognized form of brain injury in SCD. SCI is associated with decreased neurocognitive function and increased risk for new or enlarging SCI/stroke. The purpose of this study was to use mass spectrometry (MS) based proteomics to discover and validate plasma brain proteins associated with brain injury in children with SCD. The plasma samples used for the proteomics discovery analyses were from two groups: screening samples of participants in the Silent Cerebral Infarct Multi-Center Clinical Trial (SIT Trial) (n=15), and 6 age-matched, healthy non-SCD controls, three of whom had sickle cell trait. Samples from children with SCD were divided into two groups: those with SCI (n=7) and those without SCI (n=8) matched for age, hemoglobin (Hb) and white blood cell counts. To identify circulating markers of SCI, plasma proteomes were analyzed using a sequential separation approach of Hb and top abundant plasma protein depletion, followed by reverse phase separation of intact proteins, trypsin digestion and tandem MS. Neurogranin (NRGN), a small calmodulin-binding synaptic protein (PMID 9886843), was the most abundant brain-enriched protein found in the plasma of children with SCD. We developed a NRGN sandwich immunoassay for verification of MS analysis results in an independent cohort from the SIT Trial longitudinal samples. For this verification study, plasma samples from the SIT Trial and 25 normal controls were tested for NRGN levels at study entry and exit by immunoassay. The majority of the SCD samples were obtained from an ancillary study to collect longitudinal samples that started after the SIT Trial had begun. There was no overlap between the discovery and the verification group samples. The normal control samples were from 25 healthy, age and gender comparable non-SCD pediatric controls unrelated to the SIT Trial, without evidence of acute/chronic illness. From the discovery SCD group, 1172 unambiguous proteins were identified; 639 proteins were identified from normal controls, as shown in Figure 1. Twenty-five percent (289/1172) of these proteins were found solely in the SCI group. Our MS protein identification data were filtered against this list of expressed brain proteins to produce a composite list of brain proteins found in plasma from children with SCD, but not found in plasma from age, gender and race-matched healthy control children. Twenty-five plasma proteins known to be expressed in one or more cell type enriched in the human brain based on bioinformatics searches were more abundant in the SCD vs control groups, as shown in Table 1. Median NRGN levels were higher at study entry in children with SCD (0.44 ng/mL, N=98), in comparison to a healthy, non-SCD control group (0.12 ng/mL, N=25, p &lt; 0.0001), as shown in Figure 2. Using the longitudinal samples from the SIT Trial, there were no differences in NRGN levels between the observation group and the treatment group (p=0.35) over time. There was no significant difference in median NRGN levels between the observation group and the treatment group at the initial visit (0.36 vs 0.19, p=0.069) or final visit (0.39 vs 0.52, p=0.52). Using the initial visit samples from the SIT Trial, there was no significant difference in median NRGN levels between the non-SCI (n=35) and SCI groups (n=61) (p=0.073). There was no significant correlation between NRGN levels and neuropsychological measures of executive function (rho=0.089, p=0.58) and IQ (rho=-0.081, p=0.62). Our non-biased proteomic discovery studies demonstrate that NRGN levels are increased in children with SCD, when compared to normal children. Additional studies will be necessary to determine whether NRGN levels correlate with specific forms of neurologic injury. Further validation studies of larger cohorts with a multi-analyte ELISA of these brain proteins identified in plasma (Table 1) are warranted, and could help establish possible links between brain-specific proteins and neurocognitive outcomes. Disclosures Lance: NIH: Research Funding; KKI: Research Funding. Faulcon:FDA: Employment, Other: Although the author is a FDA/CTP employee, this work was not done as part his/her official duties. This publication reflects the views of the author and should not be construed to reflect the FDA/CTP's views or policies.. Fu:GSK: Employment. Yang:ImmunArray: Patents &amp; Royalties: ImmunArray. Strouse:Global Blood Therapeutics: Consultancy. Barron-Casella:Mast Pharmaceudicals - spouse: Consultancy; ImmunArray - spouse: Patents &amp; Royalties. Van Eck:ImmunArray: Consultancy, Patents &amp; Royalties. Casella:NIH: Research Funding; Mast Pharmaceuticals: Consultancy; Immunoarray Ltd.: Patents &amp; Royalties. Everett:ImmunArray: Consultancy, Patents &amp; Royalties.

Paper-based cascade cationic isotachophoresis: Multiplex detection of cardiac markers

Paper-based analytical devices (PADs) are widely used in point-of-care testing (POCT) as they are cost-effective, simple and straightforward. However, poor sensitivity hinders their use in detecting diseases with low abundance biomarkers. The poor detection limit of PADs is mainly attributed to the low concentration of analytes, and the complexity of biological fluid, leading to insufficient interactions between analytes and capture antibodies. This study aims to overcome these difficulties by developing a paper-based cationic isotachophoresis (ITP) approach for simultaneously detecting pico-molar levels of two essential cardiac protein markers: acidic troponin T (cTnT) and basic troponin I (cTnI) spiked into human serum samples. The approach utilizes 3-aminopropyltrimethoxysilane (APTMS) treated glass fiber papers with decreasing cross-sectional area assembled on a 3D printed cartridge device. Our results showed that in the presence of cTnT monoclonal antibody (mAb), fluorescently labeled cTnI and cTnT could be effectively enriched in cationic ITP. Each individual target was captured subsequently by a test line in the detection zone where the capture mAb was immobilized. Detailed analysis suggests that the technology is capable of simultaneous on-board depletion of abundant plasma proteins and enrichment of cTnI/cTnT by ~1300-fold with a sensitivity of 0.6 pmol/L for cTnT and a sensitivity of 1.5 pmol/L for cTnI in less than 6 min. The results demonstrate the potential of this technology for rapid, ultra-sensitive and cost-effective analysis of multiplex protein markers in clinical serum samples at point of care.

Abundant plasma protein depletion using ammonium sulfate precipitation and Protein A affinity chromatography

Plasma is a highly valuable resource for biomarker research since it is easy obtainable and contains a high amount of information on patient health status. Although advancements in the field of proteomics enabled analysis of the plasma proteome, identification of low abundant proteins remains challenging due to high complexity and large dynamic range. In order to reduce the dynamic range of protein concentrations, a tandem depletion technique consisting of ammonium sulfate precipitation and Protein A affinity chromatography was developed. Using this method, 50% of albumin, together with other high abundant proteins such as alpha-1-antitrypsin, was depleted from the plasma sample at 20% to 40% ammonium sulfate saturation levels. In combination with immunoglobulin removal using a Protein A column, this technique delivered up to 40 new low- to medium abundance protein identifications when performing a shotgun mass spectrometry analysis. Compared to non-depleted plasma, 270 additional protein spots were observed during 2D-PAGE analysis. These results illustrate that this tandem depletion method is equivalent to commercial kits which are based on immune-affinity chromatography. Moreover, this method using Protein A immunoglobulin depletion was shown to be highly reproducible and a minimal amount of non-target proteins was depleted. The combination of ammonium sulfate precipitation and Protein A affinity chromatography offers a low cost, efficient, straightforward and reproducible alternative to commercial kits, with proteins remaining in native conformation, allowing protein activity and protein interaction studies.

Distinct protein signature of hypertension-induced damage in the renal proteome of the two-kidney, one-clip rat model.

Hypertensive nephrosclerosis is one of the most frequent causes of chronic kidney failure. Proteome analysis potentially improves the pathophysiological understanding and diagnostic precision of this disorder. In the present exploratory study, we investigated experimental nephrosclerosis in the two-kidney, one-clip (2K1C) hypertensive rat model. The renal cortex proteome from juxtamedullary cortex and outer cortex of 2K1C male Wistar-Hannover rats (n = 4) was compared with the sham-operated controls (n = 6), using mass spectrometry-based quantitative proteomics. We combined a high abundant plasma protein depletion strategy with an extended liquid chromatographic gradient to improve peptide and protein identification. Immunohistology was used for independent confirmation of abundance. We identified 1724 proteins, of which 1434 were quantified with at least two unique peptides. Comparative proteomics revealed 608 proteins, including the platelet-derived growth factor receptor-β signalling pathway, with different abundances between the non-clipped kidney of hypertensive 2K1C rats and the corresponding kidney of the normotensive controls (P < 0.05, absolute fold change ≥1.5). Among the most significantly altered proteins in the whole cortex were periostin, transgelin, and creatine kinase B-type. Relative abundance of periostin alone allowed clear classification of 2K1C and controls. Enrichment of periostin in 2K1C rats was verified by immunohistology, showing positivity especially around the fibrotic vessels. The proteome is altered in hypertension-induced kidney damage. We propose periostin, especially in combination with transgelin and creatine kinase B-type, as possible proteomic classifier to distinguish hypertensive nephrosclerosis from the normal tissue. This classifier needs to be further validated with respect to early diagnosis of fibrosis, prognosis, and its potential as a novel molecular target for pharmacological interventions.

Performance evaluation of affinity ligands for depletion of abundant plasma proteins

Human plasma is a commonly used diagnostic fluid in clinical chemistry. In-depth plasma proteomic analysis is performed to search for disease biomarkers, however the large dynamic range of protein abundance in plasma presents a substantial analytical challenge. Removal of abundant plasma proteins using antibody capture approaches is a common and attractive means to reduce sample complexity and to aid the analysis of lower abundance proteins of interest. A novel class of heavy chain camelid-derived affinity ligands produced in Saccharomyces cerevisiae, has recently been developed as an alternative to antibody-based depletion methods. Here, we evaluate the performance characteristics of these ligands for removal of high abundance plasma proteins. Affinity ligands were tested for the removal of 14 abundant human plasma proteins. The performance characteristics were evaluated by gel-electrophoresis and LC-MS/MS of the bound and flow-through fractions. The capacity of a 5.6mL column was found to be 125μL of plasma. Replicate analysis demonstrated high column reproducibility and linearity, efficient removal of abundant proteins, and enrichment of lower abundance proteins observed after depletion. The novel class of affinity ligands provides an attractive alternative to traditional antibody-based immunodepletion methods.

Potential mechanisms of the acute coronary syndrome presentation in patients with the coronary slow flow phenomenon — Insight from a plasma proteomic approach

AimsThe coronary slow flow phenomenon [CSFP] is a coronary microvascular disorder, characterized by delayed distal vessel opacification despite the absence of obstructive coronary artery disease. Patients frequently present with an acute coronary syndrome [ACS] although the pathophysiological mechanisms responsible are unknown. The aim of this study was to identify potential mechanisms for the ACS presentation associated with the CSFP using a plasma proteomic profiling approach. Methods and resultsPlasma samples from nine CSFP subjects [56±11years] were assayed for high sensitivity C-reactive protein [hsCRP], troponin T [TnT], creatine kinase [CK], and proteomic analyses (n=6), during an ACS presentation and one month later [chronic phase]. Proteomic analysis involved chromatographic depletion of abundant plasma proteins followed by two-dimensional differential gel electrophoresis [2-D DIGE]. Protein spots demonstrating ±1.5-fold change relative to the control were identified by mass spectrometry and two differentially expressed proteins were selected for validation via Western blotting. During the ACS presentation, hsCRP was elevated [ACS=14.9±3.9mg/L vs chronic=4.23±1.37mg/L, p=0.05] but TnT and CK levels were unchanged. Proteomic analysis identified six proteins that were significantly different in abundance between the acute and chronic samples. During the ACS presentation there was a 1.6±0.13 fold increase in the anti-oxidant enzyme paraoxonase-1 and an increase in inflammatory proteins alpha-1-antichymotrypsin [1.65±0.13 fold] and alpha-1-antitrypsin [2.5±0.34 fold]. The latter was confirmed by Western blotting [1.33±0.17 OD acute/chronic ratio, p=0.05]. ConclusionThe findings from this novel detailed approach, implicate an inflammatory/oxidative stress process in the pathogenesis of the ACS presentation associated with the CSFP. Future studies should further elucidate these mechanisms.

Depletion of Abundant Plasma Proteins and Limitations of Plasma Proteomics

Immunoaffinity depletion with antibodies to the top 7 or top 14 high-abundance plasma proteins is used to enhance detection of lower abundance proteins in both shotgun and targeted proteomic analyses. We evaluated the effects of top 7/top 14 immunodepletion on the shotgun proteomic analysis of human plasma. Our goal was to evaluate the impact of immunodepletion on detection of proteins across detectable ranges of abundance. The depletion columns afforded highly repeatable and efficient plasma protein fractionation. Relatively few nontargeted proteins were captured by the depletion columns. Analyses of unfractionated and immunodepleted plasma by peptide isoelectric focusing (IEF), followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), demonstrated enrichment of nontargeted plasma proteins by an average of 4-fold, as assessed by MS/MS spectral counting. Either top 7 or top 14 immunodepletion resulted in a 25% increase in identified proteins compared to unfractionated plasma. Although 23 low-abundance (<10 ng mL(-1)) plasma proteins were detected, they accounted for only 5-6% of total protein identifications in immunodepleted plasma. In both unfractionated and immunodepleted plasma, the 50 most abundant plasma proteins accounted for 90% of cumulative spectral counts and precursor ion intensities, leaving little capacity to sample lower abundance proteins. Untargeted proteomic analyses using current LC-MS/MS platforms-even with immunodepletion-cannot be expected to efficiently discover low-abundance, disease-specific biomarkers in plasma.

Antigen Identification and Characterization of Lung Cancer Specific Monoclonal Antibodies Produced by mAb Proteomics

A mass spectrometric (MS)-based strategy for antigen (Ag) identification and characterization of globally produced monoclonal antibodies (mAbs) is described. Mice were immunized with a mixture of native glycoproteins, isolated from the pooled plasma of patients with nonsmall cell lung cancer (NSCLC), to generate a library of IgG-secreting hybridomas. Prior to immunization, the pooled NSCLC plasma was subjected to 3-sequential steps of affinity fractionation, including high abundant plasma protein depletion, glycoprotein enrichment, and polyclonal antibody affinity chromatography normalization. In this paper, to demonstrate the high quality of the globally produced mAbs, we selected 3 mAbs of high differentiating power against a matched, pooled normal plasma sample. After production of large quantities of the mAbs from ascites fluids, Ag identification was achieved by immunoaffinity purification, SDS-PAGE, Western blotting, and MS analysis of in-gel digest products. One antigen was found to be complement factor H, and the other two were mapped to different subunits of haptoglobin (Hpt). The 2 Hpt mAbs were characterized in detail to assess the quality of the mAbs produced by the global strategy. The affinity of one of the mAbs to the Hpt native tetramer form was found to have a K(D) of roughly 10(-9) M and to be 2 orders of magnitude lower than the reduced form, demonstrating the power of the mAb proteomics technology in generating mAbs to the natural form of the proteins in blood. The binding of this mAb to the beta-chain of haptoglobin was also dependent on glycosylation on this chain. The characterization of mAbs in this work reveals that the global mAb proteomics process can generate high-quality lung cancer specific mAbs capable of recognizing proteins in their native state.

The effects of abundant plasma protein depletion on global glycan profiling using nanoLC FT-ICR mass spectrometry.

We report the results of abundant plasma protein depletion on the analysis of underivatized N-linked glycans derived from plasma proteins by nanoLC Fourier-transform ion cyclotron resonance mass spectrometry. N-linked glycan profiles were compared between plasma samples where the six most abundant plasma proteins were depleted (n = 3) through a solid-phase immunoaffinity column and undepleted plasma samples (n = 3). Three exogenous glycan standards were spiked into all samples which allowed for normalization of the N-glycan abundances. The abundances of 20 glycans varying in type, structure, composition, and molecular weight (1,200-3,700 Da) were compared between the two sets of samples. Small fucosylated non-sialylated complex glycans were found to decrease in abundance in the depleted samples (greater than or equal to tenfold) relative to the undepleted samples. Protein depletion was found to marginally effect (less than threefold) the abundance of high mannose, hybrid, and large highly sialylated complex species. The significance of these findings in terms of future biomarker discovery experiments via global glycan profiling is discussed.

Effect of Plasma Protein Depletion on BNP-32 Recovery

Depletion of abundant proteins from plasma and serum is an important initial step in many biomarker discovery platforms(1). Decreasing the concentrations of highly abundant proteins (e.g., albumin, IgG, and antitrypsin) facilitates the use of contemporary proteomics technologies, such as gel electrophoresis and mass spectrometry, for detection and identification of low-abundant proteins. Furthermore, decreasing abundant protein concentrations may also improve immunoprecipitation recovery efficiencies for targeted low-abundant species by decreasing nonspecific binding (i.e., shielding the antigen-binding domain) to the antibody and/or solid supports. A notable pitfall to depletion strategies is the potential for unintentionally removing low-abundant plasma or serum proteins. These low-abundant species may be bound specifically or nonspecifically to the depletion ligand, depletion target protein (e.g., carrier proteins), or the solid support(s). Thus, it is important to critically evaluate the effectiveness of abundant plasma protein depletion for enhancing the study of low-abundant protein biomarker(s). We have been actively developing a targeted biomarker discovery platform for characterizing the circulating forms of B-type natriuretic peptide (BNP) that includes protein depletion strategies, immunoprecipitation, gel electrophoresis, isotope dilution (absolute quantification), and nanoflow liquid chromatography coupled to high-performance hybrid Fourier transform mass …

Rat plasma proteomics: Effects of abundant protein depletion on proteomic analysis

The proteomic analysis of plasma and serum samples represents a formidable challenge due to the presence of a few highly abundant proteins such as albumin and immunoglobulins. Detection of low abundance protein biomarkers requires therefore either the specific depletion of high abundance proteins with immunoaffinity columns and/or optimized protein fractionation methods based on charge, size or hydrophobicity. Here we describe the depletion of seven abundant rat plasma proteins with an immunoaffinity column with coupled antibodies directed against albumin, IgG, transferrin, IgM, haptoglobin, fibrinogen and α1-anti-trypsin. The IgY-R7-LC2 (Beckman Coulter) column showed high specificity for the targeted proteins and was able to efficiently remove most of the albumin, IgG and transferrin from rat plasma samples as judged by Western blot analysis. Depleted rat plasma protein samples were analyzed by SELDI-TOF MS, 2 D SDS-PAGE and 2D-LC and compared to non-depleted plasma samples as well as to the abundant protein fraction that was eluted from the immunoaffinity column. Analysis of the depleted plasma protein fraction revealed improved signal to noise ratios, regardless of which proteomic method was applied. However, only a small number of new proteins were observed in the depleted protein fraction. Immunoaffinity depletion of abundant plasma proteins results in the significant dilution of the original sample which complicates subsequent analysis. Most proteomic approaches require specialized sample preparation procedures during which significant losses of less abundant proteins and potential biomarkers can occur. Even though abundant protein depletion reduces the dynamic range of the plasma proteome by about 2–3 orders of magnitude, the difference between medium-abundant and low abundant plasma proteins is still in the range of 7–8 orders of magnitude and beyond the dynamic range of current proteomic technologies. Thus, exploring the plasma proteome in greater detail remains a daunting task.