Enrichment of serum low-molecular-weight proteins using C18 absorbent under urea/dithiothreitol denatured environment (original) (raw)
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ELECTROPHORESIS, 2005
We present a simple protocol for affinity depletion to remove the two most abundant serum proteins, albumin and immunoglobulin G (IgG). Under native conditions, albumin/IgG were efficiently removed and several proteins were enriched as shown by two-dimensional electrophoresis (2-DE). Besides that, partly denaturing conditions were established by adding 5 or 20% acetonitrile (ACN) in order to disrupt the binding of low-molecular-weight (LMW) proteins to the carrier proteins albumin/IgG. 2-DE results showed that the total number of detected LMW proteins increased under denaturing conditions when compared to native conditions. Interestingly, the presence of 5% ACN in serum revealed better enrichment of LMW proteins when compared to 20% ACN condition. Seven randomly distributed spots in albumin/IgG depleted serum samples under 5% ACN condition were picked from the 2-DE gels and identified by mass spectrometry (MS). The intensity of five LMW protein spots increased under denaturing conditions when compared to native conditions. Three of the seven identified spots (serum amyloid P, vitamin D-binding protein, and transthyretin) belong to a group of relatively low-abundant proteins, which make up only 1% of all serum proteins. The method presented here improves the resolution of the serum proteome by increasing the number of visualized spots on 2-D gels and allowing the detection and MS identification of LMW proteins and proteins of lower abundance.
Molecular & Cellular Proteomics, 2007
Biomarker discovery produces lists of candidate markers whose presence and level must be subsequently verified in serum or plasma. Verification represents a paradigm shift from unbiased discovery approaches to targeted, hypothesis-driven methods and relies upon specific, quantitative assays optimized for the selective detection of target proteins. Many protein biomarkers of clinical currency are present at or below the nanogram/milliliter range in plasma and have been inaccessible to date by MS-based methods. Using multiple reaction monitoring coupled with stable isotope dilution mass spectrometry, we describe here the development of quantitative, multiplexed assays for six proteins in plasma that achieve limits of quantitation in the 1-10 ng/ml range with percent coefficients of variation from 3 to 15% without immunoaffinity enrichment of either proteins or peptides. Sample processing methods with sufficient throughput, recovery, and reproducibility to enable robust detection and quantitation of candidate biomarker proteins were developed and optimized by addition of exogenous proteins to immunoaffinity depleted plasma from a healthy donor. Quantitative multiple reaction monitoring assays were designed and optimized for signature peptides derived from the test proteins. Based upon calibration curves using known concentrations of spiked protein in plasma, we determined that each target protein had at least one signature peptide with a limit of quantitation in the 1-10 ng/ml range and linearity typically over 2 orders of magnitude in the measurement range of interest. Limits of detection were frequently in the high picogram/milliliter range. These levels of assay performance represent up to a 1000-fold improvement compared with direct analysis of proteins in plasma by MS and were achieved by simple, robust sample processing involving abundant protein depletion and minimal fractionation by strong cation exchange chromatography at the peptide level prior to LC-multiple reaction monitoring/MS. The methods presented here provide a solid basis for developing quantitative MS-based assays of low level proteins in blood.
International Journal of Molecular Sciences, 2020
In the present study, we evaluated four small molecule affinity-based probes based on agarose-immobilized benzamidine (ABA), O-Phospho-L-Tyrosine (pTYR), 8-Amino-hexyl-cAMP (cAMP), or 8-Amino-hexyl-ATP (ATP) for their ability to remove high-abundant proteins such as serum albumin from plasma samples thereby enabling the detection of medium-to-low abundant proteins in plasma samples by mass spectrometry-based proteomics. We compared their performance with the most commonly used immunodepletion method, the Multi Affinity Removal System Human 14 (MARS14) targeting the top 14 most abundant plasma proteins and also the ProteoMiner protein equalization method by label-free quantitative liquid chromatography tandem mass spectrometry (LC-MSMS) analysis. The affinity-based probes demonstrated a high reproducibility for low-abundant plasma proteins, down to picomol per mL levels, compared to the Multi Affinity Removal System (MARS) 14 and the Proteominer methods, and also demonstrated superio...
Analysis of low-abundance, low-molecular-weight serum proteins using mass spectrometry
Journal of biomolecular techniques : JBT, 2004
To detect diseases early in the general population, new diagnostic approaches are needed that have adequate sensitivity and specificity. Recent studies have used mass spectrometry to identify a serum proteomic pattern for breast and ovarian cancer. Serum contains 60-80 mg/mL protein, but 57-71% of this is serum albumin, and 8-26% are gamma-globulins. These large proteins must be depleted before smaller, less-abundant proteins can be detected using mass spectrometry, but because serum albumin is known to act as a carrier for smaller proteins, removal of these molecules using columns or filtration may result in the loss of molecules of interest. The objective of this study was to develop a reproducible method to deplete serum samples of high-abundance proteins in order to analyze the less-abundant proteins present in serum. We used organic solvents to precipitate the large proteins out of solution. We also predicted that this would cause many smaller proteins to dissociate from their ...
Proceedings of the National Academy of Sciences, 2012
Sensitive detection of low-abundance proteins in complex biological samples has typically been achieved by immunoassays that use antibodies specific to target proteins; however, de novo development of antibodies is associated with high costs, long development lead times, and high failure rates. To address these challenges, we developed an antibody-free strategy that involves PRISM (high-pressure, high-resolution separations coupled with intelligent selection and multiplexing) for sensitive selected reaction monitoring (SRM)-based targeted protein quantification. The strategy capitalizes on high-resolution reversed-phase liquid chromatographic separations for analyte enrichment, intelligent selection of target fractions via on-line SRM monitoring of internal standards, and fraction multiplexing before nano-liquid chromatography-SRM quantification. Application of this strategy to human plasma/serum demonstrated accurate and reproducible quantification of proteins at concentrations in the 50-100 pg/mL range, which represents a major advance in the sensitivity of targeted protein quantification without the need for specific-affinity reagents. Application to a set of clinical serum samples illustrated an excellent correlation between the results obtained from the PRISM-SRM assay and those from clinical immunoassay for the prostate-specific antigen level.
Journal of Proteome Research, 2005
Today, proteomics is an exciting approach to discover potential biomarkers of different disorders. One challenge with proteomics experiments is the wide concentration range of proteins in various tissues and body fluids. The most abundant component in human body fluids, human serum albumin (HSA), is present at concentrations corresponding to approximately 50% of the total protein content in, e.g., plasma and cerebrospinal fluid (CSF). If this component could be selectively removed, then the chances of observing lower-abundance component of clinical interest would be greatly improved. There are today several approaches of varying specificity available for depletion. In this study, the properties of two commercially available kits, for the removal of HSA and HSA and immunoglobulin G (IgG), respectively, were compared, and the benefits of using depletion steps prior to on-line LC-FTICR MS were evaluated. Both methods were applied on plasma and CSF. To our knowledge, these are the first results reported for CSF. Also, the combination with electrospray LC-FTICR MS is novel. The proportion of depleted HSA and IgG was estimated using global labeling markers for peptide quantification. Both depletion-methods provided a significant reduction of HSA, and the identification of lower abundant components was clearly facilitated. A higher proportion of HSA was removed using the affinity-based removal kit, and consequently more proteins could be identified using this approach. a The depletion step was repeated for three aliquots of each sample, except for plasma treated with the HSA-depletion kit, where two samples were prepared. The variation in total protein concentration is <13% for all depleted samples.
Proteomics Clinical Applications, 2007
The low molecular weight (LMW) region of the circulatory proteome, thought to contain a rich source of biomarkers, resides in vivo, in a complexed state with larger, highly abundant resident proteins. Consequently, serum fractionation approaches that deplete the high-abundance proteins under native conditions will remove much of the LMW proteome. We describe a new strategy to systematically collect, isolate and enrich the LMW molecules that would be otherwise eliminated during the depletion of high-abundance circulatory proteins based on continuous elution electrophoresis. We employ strong denaturing conditions to disrupt association with the high-abundance carrier proteins followed by fractionation and removal of SDS. Under denaturation, the LMW molecules were effectively stripped from the highly abundant carrier proteins. We then removed the SDS by ion exchange matrix sequestration and concentrated the fractions. The outcome is a series of SDS-free fractions of LMW molecules. The isolated fractions were then analyzed by enzymatic digestion followed by LC-MS/MS analysis. The yield of multiple peptide hits as well as the total number of identifications significantly increased (50%) compared to unfractionated serum. The method yielded a 30% higher number of low-abundance serum proteins compared to direct sequencing of unfractionated serum.
Targeted Quantification of Low ng/mL Level Proteins in Human Serum without Immunoaffinity Depletion
Journal of Proteome Research, 2013
We recently reported an antibody-free targeted protein quantification strategy, termed high-pressure, high-resolution separations with intelligent selection and multiplexing (PRISM), for achieving significantly enhanced sensitivity using selected reaction monitoring (SRM) mass spectrometry. Integrating PRISM with front-end IgY14 immunoaffinity depletion, sensitive detection of targeted proteins at 50−100 pg/mL levels in human blood plasma/serum was demonstrated. However, immunoaffinity depletion is often associated with undesired losses of target proteins of interest. Herein we report further evaluation of PRISM-SRM quantification of low-abundance serum proteins without immunoaffinity depletion. Limits of quantification (LOQ) at low ng/mL levels with a median coefficient of variation (CV) of ∼12% were achieved for proteins spiked into human female serum. PRISM-SRM provided >100-fold improvement in the LOQ when compared to conventional LC-SRM measurements. PRISM-SRM was then applied to measure several low-abundance endogenous serum proteins, including prostate-specific antigen (PSA), in clinical prostate cancer patient sera. PRISM-SRM enabled confident detection of all target endogenous serum proteins except the low pg/mL-level cardiac troponin T. A correlation coefficient >0.99 was observed for PSA between the results from PRISM-SRM and immunoassays. Our results demonstrate that PRISM-SRM can successfully quantify low ng/mL proteins in human plasma or serum without depletion. We anticipate broad applications for PRISM-SRM quantification of low-abundance proteins in candidate biomarker verification and systems biology studies.
Quantitative assessment of human serum high-abundance protein depletion
ELECTROPHORESIS, 2008
The aim of this study is to quantify the effectivity of the depletion of human highabundance serum and plasma proteins for improved protein identification and disease marker candidate discovery and to assess the risk of concomitant removal of relevant marker proteins. 2-DE and bottom-up shotgun MS combining 2-D capillary chromatography with MS/MS were applied in parallel for the analysis of fractions resulting from the depletion procedure. For many proteins the factors of enrichment by the depletion were obvious allowing their enhanced detection and identification upon high-abundance protein depletion. Nano-liquid chromatography linked MS allowed the efficient identification of several low-abundant proteins that were not identified on the 2-DE gels. Resolving the fractions that were eluted from the matrix upon depletion indicated unspecific binding of disease relevant proteins in plasma samples from acute myocardial infarction patients. The unspecific binding to the depletion matrix of inflammatory markers spiked into the serum was found to depend on the type of capturing agent used. Polyclonal avian antibodies (IgY) displayed the least unspecific binding due to the high immunogenicity of mammalian proteins in avian hosts.
Journal of Chromatography A, 2009
Human serum albumin (HSA) and immunoglobulin G (IgG) represent over 75% of all proteins present in human plasma. These high-abundance proteins prevent the detection of low-abundance proteins which are potential markers for various diseases. The depletion of HSA and IgG is therefore essential for further proteome analysis. In this paper we describe the optimization of conditions for selective depletion of HSA and IgG using affinity and pseudo-affinity chromatography. A BIA Separations CIM (convective interaction media) Protein G disk was applied for the removal of IgG and the Mimetic Blue SA A6XL stationary phase for the removal of HSA. The binding and the elution buffer for CIM Protein G disk were chosen on the basis of the peak shape. The dynamic binding capacity was determined. It was shown to be dependent on the buffer system used and independent of the flow rate and of the concentration of IgG. Beside the binding capacity for the IgG standard, the binding capacity was also determined for IgG in human plasma. The Mimetic Blue SA A6XL column was characterized using human plasma. The selectivity of the depletion was dependent on the amount of human plasma that was loaded on the column. After the conditions on both supports had been optimized, the Mimetic Blue SA A6XL stationary phase was combined with the CIM Protein G disk in order to simultaneously deplete samples of human plasma. A centrifuge spin column that enables the removal of IgG and HSA from 20 L of human plasma was designed. The results of the depletion were examined using sodium dodecyl sulfate polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis.