Enrichment Of Low-abundance Proteins Research Articles

Expect the Unexpected Enrichment of "Hidden Proteome" of Seeds and Tubers by Depletion of Storage Proteins.

Dynamic resolution of seed and tuber protein samples is highly limited due to the presence of high-abundance storage proteins (SPs). These proteins inevitably obscure the low-abundance proteins (LAPs) impeding their identification and characterization. To facilitate the detection of LAPs, several methods have been developed during the past decade, enriching the proteome with extreme proteins. Most of these methods, if not all, are based on the specific removal of SPs which ultimately magnify the proteome coverage. In this mini-review, we summarize the available methods that have been developed over the years for the enrichment of LAPs either from seeds or tubers. Incorporation of these methods during the protein extraction step will be helpful in understanding the seed/tuber biology in greater detail.

A Chemical Glycoproteomics Platform To study the Human Prostate Secretome

Accurate diagnosis and treatment of prostate cancer demands a highly specific biomarker of both the disease, progression and prognosis. As glycoproteins have proven to be altered in most cancers, we have focused on studying glycoproteins in human prostate tissue samples. We have developed an efficient method for labeling and enriching glycoproteins, followed by MS‐based analysis of normal and carcinoma tissue. We sought to selectively enrich secreted glycoproteins because of the potential of secreted glycoprotein biomarkers in non‐invasive prostate cancer detection. This is accomplished by culturing human prostate tissue derived from radical prostatectomies, at varying states of prostatic disease, and collecting the cultured media which samples secretome proteins. During culturing, tissue is chemically tagged with an azide functionalized monosaccharide, N‐azidoacetylmannosamine‐tetraacylated (ManNAz). Labeled proteins are subsequently reacted with a biotin‐functionalized probe for the capture of azido‐sialylated glycoproteins. After analysis of confirmed azide‐dependent labeling, glycoproteins were digested and analyzed on a linear ion trap mass spectrometer with collision‐induced dissociation (CID). Greater than 80% of the proteins identified by LC‐MS/MS were secreted proteins, indicating that this strategy achieves enrichment of low abundance proteins. Additionally, a comparative proteomics analysis between diseased and healthy tissue identified proteins unique to each tissue type, which are undergoing validation as potential biomarkers. Virtually all clinically approved cancer biomarkers are cell‐surface or secreted glycoproteins and this work extends the application of existing metabolic labeling techniques, while developing new methodologies for cell surface labeling to discover biomarkers associated with prostate cancer.Support or Funding InformationThis work was funded by grants from the NIH to C.R.B. (GM066047 and GM058867) and to S.L.M. (2T32DK007217‐37). D.R.S. is supported by NSF predoctoral fellowships.

Effects of ultrasonic treatment on removal of abundant proteins and enrichment of low-abundance proteins in defatted soybean meal by isopropanol

ABSTRACTThe establishment of a method for the removal of abundant proteins (APs) and enrichment of low-abundance proteins (LAPs) might contribute to further studies on the physiological functions of LAPs in animals and humans without the influence of APs. The aim of our study was to investigate the effect of ultrasonic treatment on the removal of APs and enrichment of LAPs in defatted soybean meal using isopropanol. The optimal isopropanol concentration for the removal APs was initially shown to be 50% without ultrasonic treatment. The highest protein concentration was achieved at 50% isopropanol concentration with 1 h of total extraction time (ultrasonic treatment and water-bath vibrating) and when the sample was treated with ultrasound at 400 W for 10 min. Under these conditions, the protein concentration increased by 26.42% when compared to the control (no ultrasonic treatment). Furthermore, no β-conglycinin and glycinin was detected by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and mass spectrometry.

Proteomic Analysis of Lonicera japonica Thunb. Immature Flower Buds Using Combinatorial Peptide Ligand Libraries and Polyethylene Glycol Fractionation.

Lonicera japonica Thunb. flower is a well-known medicinal plant that has been widely used for the treatment of human disease. To explore the molecular mechanisms underlying the biological activities of L. japonica immature flower buds, a gel-free/label-free proteomic technique was used in combination with combinatorial peptide ligand libraries (CPLL) and polyethylene glycol (PEG) fractionation for the enrichment of low-abundance proteins and removal of high-abundance proteins, respectively. A total of 177, 614, and 529 proteins were identified in crude protein extraction, CPLL fractions, and PEG fractions, respectively. Among the identified proteins, 283 and 239 proteins were specifically identified by the CPLL and PEG methods, respectively. In particular, proteins related to the oxidative pentose phosphate pathway, signaling, hormone metabolism, and transport were highly enriched by CPLL and PEG fractionation compared to crude protein extraction. A total of 28 secondary metabolism-related proteins and 25 metabolites were identified in L. japonica immature flower buds. To determine the specificity of the identified proteins and metabolites for L. japonica immature flower buds, Cerasus flower buds were used, which resulted in the abundance of hydroxymethylbutenyl 4-diphosphate synthase in L. japonica immature flower buds being 10-fold higher than that in Cerasus flower buds. These results suggest that proteins related to secondary metabolism might be responsible for the biological activities of L. japonica immature flower buds.

Array-Based Two Dimensional Liquid Chromatography System for Proteomic Analysis of Human Plasma

Abstract The human plasma proteome has the characteristics of complexity in component and large dynamic range of protein concentrations. Herein, an array-based online two dimensional liquid chromatography system combined with protein equalizer technology was developed for the large-scale depletion of high abundance proteins and enrichment of low abundance proteins in human plasma. The array-based online two dimensional liquid chromatography system could be used to separate the plasma at the intact protein level with good reproducibility and high throughput. The total separation time was only 4 h and the fast location of high abundance proteins was also achieved. After the high abundance protein fractions was treated by ampholine@PM polymer microsphere, the number of identified low abundance proteins increased ten-fold, which significantly decreased the loss of low abundance proteins in high abundance protein fractions. The techniques combined were then applied to perform the proteomic analysis of human plasma sample. The total number of identified proteins was 1474 and the dynamic range of protein concentration was 7. In this work, 252 proteins were identified in high abundance protein fractions, among which 61 proteins belonged to high abundance proteins. These results demonstrated that an array-based online two dimensional liquid chromatography system combined with protein equalizer technology could efficiently achieve the large-scale depletion of high abundance proteins and the enrichment of low abundance proteins in human plasma, with a remarkable improvement in protein identification and a great prospect in the proteomic research of other complex samples.

Comparison of Depletion Strategies for the Enrichment of Low-Abundance Proteins in Urine.

Proteome analysis of complex biological samples for biomarker identification remains challenging, among others due to the extended range of protein concentrations. High-abundance proteins like albumin or IgG of plasma and urine, may interfere with the detection of potential disease biomarkers. Currently, several options are available for the depletion of abundant proteins in plasma. However, the applicability of these methods in urine has not been thoroughly investigated. In this study, we compared different, commercially available immunodepletion and ion-exchange based approaches on urine samples from both healthy subjects and CKD patients, for their reproducibility and efficiency in protein depletion. A starting urine volume of 500 μL was used to simulate conditions of a multi-institutional biomarker discovery study. All depletion approaches showed satisfactory reproducibility (n=5) in protein identification as well as protein abundance. Comparison of the depletion efficiency between the unfractionated and fractionated samples and the different depletion strategies, showed efficient depletion in all cases, with the exception of the ion-exchange kit. The depletion efficiency was found slightly higher in normal than in CKD samples and normal samples yielded more protein identifications than CKD samples when using both initial as well as corresponding depleted fractions. Along these lines, decrease in the amount of albumin and other targets as applicable, following depletion, was observed. Nevertheless, these depletion strategies did not yield a higher number of identifications in neither the urine from normal nor CKD patients. Collectively, when analyzing urine in the context of CKD biomarker identification, no added value of depletion strategies can be observed and analysis of unfractionated starting urine appears to be preferable.

A Chemical Glycoproteomics Platform Applied to Human Prostate Cancer

Accurate diagnosis and treatment of prostate cancer demands a highly specific biomarker of prostate cancer, as prostate‐specific antigen (PSA), the current clinical biomarker for prostate cancer, suffers from high false positive and high false negative rates that leave many men without a conclusive diagnosis. As glycoproteins have proven to be altered in most cancers, we have focused on studying glycoproteins in human prostate tissue samples. We have developed an efficient method for labeling and enriching glycoproteins, followed by MS‐based analysis of normal and carcinoma tissue. Human prostate tissue derived from radical prostatectomies, at varying states of prostatic disease is chemically tagged with an azide functionalized monosaccharide. Labeled tissue slices are reacted with a biotin‐functionalized probe for the capture of azido‐sialylated glycoproteins. Once analysis of labeled tissue confirmed azide‐dependent labeling, glycoproteins were digested and analyzed on a linear ion trap mass spectrometer with collision‐induced dissociation (CID). Greater than 60% of the proteins identified by LC‐MS/MS were cell‐surface or secreted proteins, indicating that this strategy achieves enrichment of low abundance proteins. Additionally, a comparative proteomics analysis between diseased and healthy tissue identified proteins unique to each tissue type, which are undergoing further validation as potential biomarkers. Virtually all clinically approved cancer biomarkers are glycoproteins and this work extends the application of existing metabolic labeling techniques, while developing new methodologies for cellular labeling to discover biomarkers associated with prostate cancer.

Chloroplast isolation and affinity chromatography for enrichment of low-abundant proteins in complex proteomes.

Detailed knowledge of the proteome is crucial to advance the biological sciences. Low-abundant proteins are of particular interest to many biologists as they include, for example those proteins involved in signal transduction. Recent technological advances resulted in a tremendous increase in protein identification sensitivity by mass spectrometry (MS). However, the dynamic range in protein abundance still forms a fundamental problem that limits the detection of low-abundant proteins in complex proteomes. These proteins will typically escape detection in shotgun MS experiments due to the presence of other proteins at an abundance several-fold higher in order of magnitude. Therefore, specific enrichment strategies are required to overcome this technical limitation of MS-based protein discovery. We have searched for novel signal transduction proteins, more specifically kinases and calcium-binding proteins, and here we describe different approaches for enrichment of these low-abundant proteins from isolated chloroplasts from pea and Arabidopsis for subsequent proteomic analysis by MS. These approaches could be extended to include other signal transduction proteins and target different organelles.

Development of an improved fractionation of the human plasma proteome by a combination of abundant proteins depletion and multi-lectin affinity chromatography.

Current analytical tools lack the required capacity to reduce the complexity of the plasma proteome and identify low-level proteins of clinical interest. Hence, the need to develop a fractionation approach to provide adequate throughput for a clinical study and minimize the loss and improve the detection of low abundance proteins. We present the development of an analytical platform that combines the depletion of 12 high abundance proteins and multi-lectin affinity chromatography (12P-M-LAC) fractionation. We validated the highly specific, stable and robust 12P-M-LAC platform using human plasma. An improved enrichment of low abundance proteins and glycoproteins with minimum sample loss was achieved demonstrating the suitability of this platform in future biomarker discovery studies.

Tools for systems biology.

Tools for systems biology.

Proteomic analysis of human plasma in chronic rheumatic mitral stenosis reveals proteins involved in the complement and coagulation cascade.

BackgroundRheumatic fever in childhood is the most common cause of Mitral Stenosis in developing countries. The disease is characterized by damaged and deformed mitral valves predisposing them to scarring and narrowing (stenosis) that results in left atrial hypertrophy followed by heart failure. Presently, echocardiography is the main imaging technique used to diagnose Mitral Stenosis. Despite the high prevalence and increased morbidity, no biochemical indicators are available for prediction, diagnosis and management of the disease. Adopting a proteomic approach to study Rheumatic Mitral Stenosis may therefore throw some light in this direction. In our study, we undertook plasma proteomics of human subjects suffering from Rheumatic Mitral Stenosis (n = 6) and Control subjects (n = 6). Six plasma samples, three each from the control and patient groups were pooled and subjected to low abundance protein enrichment. Pooled plasma samples (crude and equalized) were then subjected to in-solution trypsin digestion separately. Digests were analyzed using nano LC-MSE. Data was acquired with the Protein Lynx Global Server v2.5.2 software and searches made against reviewed Homo sapiens database (UniProtKB) for protein identification. Label-free protein quantification was performed in crude plasma only.ResultsA total of 130 proteins spanning 9–192 kDa were identified. Of these 83 proteins were common to both groups and 34 were differentially regulated. Functional annotation of overlapping and differential proteins revealed that more than 50% proteins are involved in inflammation and immune response. This was corroborated by findings from pathway analysis and histopathological studies on excised tissue sections of stenotic mitral valves. Verification of selected protein candidates by immunotechniques in crude plasma corroborated our findings from label-free protein quantification.ConclusionsWe propose that this protein profile of blood plasma, or any of the individual proteins, could serve as a focal point for future mechanistic studies on Mitral Stenosis. In addition, some of the proteins associated with this disorder may be candidate biomarkers for disease diagnosis and prognosis. Our findings might help to enrich existing knowledge on the molecular mechanisms involved in Mitral Stenosis and improve the current diagnostic tools in the long run.Electronic supplementary materialThe online version of this article (doi:10.1186/1559-0275-11-35) contains supplementary material, which is available to authorized users.

Identifying prostate cancer biomarkers by profiling glycoproteins in human prostate tissue (591.6)

Prostate‐specific antigen (PSA), the current clinical biomarker for prostate cancer, suffers from high false positive and high false negative rates that leave many men without a conclusive diagnosis. Virtually all clinically approved cancer biomarkers are cell‐surface or secreted glycoproteins. We seek to identify more specific clinical biomarkers of prostate cancer by developing an efficient method for specifically labeling and enriching glycoproteins, followed by MS‐based analysis of normal and cancerous human prostate tissue. We present a novel biomarker identification strategy where human prostate tissue derived from radical prostatectomies, at varying states of prostatic disease is chemically tagged. To identify cell‐surface or secreted glycoproteins from prostate tissue, we employ metabolic labeling, a technique in which unnatural azide or alkyne‐functionalized monosaccharides are incorporated, into cell‐surface glycoproteins via the cells’ own metabolic machinery. Tissue slices incubated with these labeled sugars are subsequently reacted with a biotin‐functionalized probe for the capture of sialylated glycoproteins. Analysis of labeled tissue by Western blot confirmed azide or alkyne‐dependent labeling. Proteins were then digested with trypsin and the resultant peptides were analyzed on a linear ion trap mass spectrometer with collision‐induced dissociation (CID). Greater than 60% of the proteins identified by LC‐MS/MS were cell‐surface or secreted proteins, indicating that this strategy achieves enrichment of low abundance proteins. Subsequently, we performed a comparative proteomics experiment between diseased and healthy tissue and identified proteins unique to each tissue type, which are undergoing further validation as potential biomarkers. This work extends the application of existing metabolic labeling techniques, while developing new methodologies for cell surface labeling to discover biomarkers associated with prostate cancer.

Organellar proteomics of embryonic stem cells.

Embryonic stem cells (ESCs) are undifferentiated cells with two common remarkable features known as self-renewal and differentiation. Proteomics plays an increasingly important role in understanding molecular mechanisms underlying self-renewal and pluripotency of ESCs and their applications in cell therapy and developmental biology studies. As the function of a protein is strongly associated with its localization in cell, a complete and accurate picture of the proteome of ESCs cannot be achieved without knowing the subcellular locations of proteins. Subcellular fractionation allows enrichment of low abundant proteins and signaling complexes and reduces the complexity of the sample. It also provided insight into tracking proteins that shuttle between different compartments. Despite the substantial interest and efforts in ESC subcellular proteomics area, progress has been relatively limited. In this review, we present an overview on current status of ESCs organelle proteomics research and discuss challenges in subcellular proteomics.

Impact of the depletion of high-abundance proteins in blood plasma/serum on the proteome of these media in growing farm animals

The aim of the study was to use hexapeptide combinatorial libraries to evaluate the influence of high-abundance protein depletion on low-abundance protein enrichment, protein concentration, and 2D gel quality in plasma and serum proteomes of growing piglets and calves. Depleted and nondepleted samples of plasma/serum were resolved using 2D electrophoresis (2DE). Bioinformatic analysis of the 2D gels showed an increased number of detected spots on each gel after depletion of excess high-abundance proteins in calf plasma (+26.46%) and in piglet serum (+49.40%). Relative expression of selected low-abundance protein spots in the blood plasma/serum samples was higher after high-abundance protein depletion. Both media showed decreased expressions of high-abundance protein spot clusters on 2D gels after treatment. The results of this study confirm both the high efficiency of high-abundance protein depletion and the increase in concentrations of low-abundance proteins on 2D gels after treatment. The method of depletion is reproducible and it may be used as an alternative to immunodepletion for preparing blood plasma/serum samples from young, growing animals for 2DE. Increased content of the low-abundance proteins on the 2D gels enables complete analysis of plasma/serum proteome and thereby allows for the discovery of novel biomarkers that might be useful in veterinary medicine.

High abundance plasma proteins depletion vs. low abundance proteins enrichment: Comparison of methods

To date, the complexity of the plasma proteome exceeds the analytical capacity of conventional approaches to isolate low abundance proteins that may prove to be informative biomarkers. Only complex multistep separations strategies have been able to detect a substantial number of low abundance proteins (<100 ng/ml). The first step of these protocols is generally the depletion of high abundance proteins by the use of immunoaffinity column or the low abundance proteins enrichment by the use of solid phase hexapeptides ligand libraries. Here we present a direct comparison of these two approaches. After the immuno-depletion of high abundance proteins or the enrichment of low abundance proteins, the plasma sample was further fractionated by SCX chromatography and analyzed by RP-LC-MS/MS with a Q-TOF mass spectrometer. The depletion of the 20 higher abundance plasma proteins allowed for the identification of a larger number of proteins (about 60% more) with respect to that obtainable after low abundance proteins enrichment. However, from a technical point of view, the enrichment approach seems to be more suitable as first stage of a complex multi-step fractionation protocol. Moreover, the 2 datasets are only partially overlapping, showing that the 2 methods are somehow complementary. Advantages and disadvantages of both methods are discussed.

Deciphering Systemic Wound Responses of the Pumpkin Extrafascicular Phloem by Metabolomics and Stable Isotope-Coded Protein Labeling

In cucurbits, phloem latex exudes from cut sieve tubes of the extrafascicular phloem (EFP), serving in defense against herbivores. We analyzed inducible defense mechanisms in the EFP of pumpkin (Cucurbita maxima) after leaf damage. As an early systemic response, wounding elicited transient accumulation of jasmonates and a decrease in exudation probably due to partial sieve tube occlusion by callose. The energy status of the EFP was enhanced as indicated by increased levels of ATP, phosphate, and intermediates of the citric acid cycle. Gas chromatography coupled to mass spectrometry also revealed that sucrose transport, gluconeogenesis/glycolysis, and amino acid metabolism were up-regulated after wounding. Combining ProteoMiner technology for the enrichment of low-abundance proteins with stable isotope-coded protein labeling, we identified 51 wound-regulated phloem proteins. Two Sucrose-Nonfermenting1-related protein kinases and a 32-kD 14-3-3 protein are candidate central regulators of stress metabolism in the EFP. Other proteins, such as the Silverleaf Whitefly-Induced Protein1, Mitogen Activated Protein Kinase6, and Heat Shock Protein81, have known defensive functions. Isotope-coded protein labeling and western-blot analyses indicated that Cyclophilin18 is a reliable marker for stress responses of the EFP. As a hint toward the induction of redox signaling, we have observed delayed oxidation-triggered polymerization of the major Phloem Protein1 (PP1) and PP2, which correlated with a decline in carbonylation of PP2. In sum, wounding triggered transient sieve tube occlusion, enhanced energy metabolism, and accumulation of defense-related proteins in the pumpkin EFP. The systemic wound response was mediated by jasmonate and redox signaling.

Validated hemoglobin‐depletion approach for red blood cell lysate proteome analysis by means of 2D PAGE and Orbitrap MS

The analysis of the cytosolic red blood cell (RBC) proteome is negatively affected by the high intracellular amount of hemoglobin complicating the detection of low-abundant cytosolic proteins. In this study, an alternative approach for the preparation of hemoglobin-depleted RBC lysates is presented, which was established in combination with downstream 2D PAGE analysis and Orbitrap MS. Hemoglobin removal was accomplished by using HemoVoid(TM) depletion reagent, which enabled a very efficient enrichment of low-abundant proteins by simultaneously reducing the hemoglobin concentration of the sample. After defining selected sample preparation protocol characteristics including specificity/selectivity, precision and linearity, a 2D reference map (pH 4-7) of the cytosolic RBC proteome was generated and a total of 189 different proteins were identified. Thus, the presented approach proved to be highly suitable to prepare reproducible high-resolution 2D protein maps of the RBC cytosol and provides a helpful tool for future studies investigating disease- or storage-induced changes of the cytosolic RBC proteome.

Separation of proteins from human plasma by sample displacement chromatography in hydrophobic interaction mode

Sample displacement chromatography (SDC) in reversed-phase and ion-exchange modes was introduced approximately 20 years ago. This method was first used for the preparative purification of peptides and proteins. Recently, SDC in ion-exchange mode was also successfully used for enrichment of low-abundance proteins from human plasma. In this paper, the use of SDC for the separation of plasma proteins in hydrophobic interaction mode is demonstrated. By use of two or more columns coupled in series during sample application, and subsequent elution of detached columns in parallel, additional separation of bound proteins was achieved. Further low-abundance, physiologically active proteins could be highly enriched and detected by ESI-MS/MS.

High confidence and sensitivity four-dimensional fractionation for human plasma proteome analysis

Reducing the complexity of plasma proteome through complex multidimensional fractionation protocols is critical for the detection of low abundance proteins that have the potential to be the most specific disease biomarkers. Therefore, we examined a four dimension profiling method, which includes low abundance protein enrichment, tryptic digestion and peptide fractionation by IEF, SCX and RP-LC. The application of peptide pI filtering as an additional criterion for the validation of the identifications allows to minimize the false discovery rate and to optimize the best settings of the protein identification database search engine. This sequential approach allows for the identification of low abundance proteins, such as angiogenin (10(-9) g/L), pigment epithelium growth factor (10(-8) g/L), hepatocyte growth factor activator (10(-7) g/L) and thrombospondin-1 (10(-6) g/L), having concentrations similar to those of many other growth factors and cytokines involved in disease pathophysiology.

On-plate enrichment methods for MALDI-MS analysis in proteomics

As one of the most comprehensive and versatile tools for investigating proteomics, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) still faces the problem of identification of low abundant proteins and post-translational modifications (PTMs) of proteins. These proteins typically require concentration and isolation prior to MS analysis. The surfaces of MALDI targets have been modified so they can be used not only as sample supports but as functional substrates for sample enrichment. These modified targets are therefore capable of concentrating and binding molecules of interest selectively from sample solutions placed on them. In this report, current on-plate techniques employed for enrichment of low-abundance proteins, including those for selective capture of phosphopeptides and glycopeptides prior to MALDI-MS analysis, were reviewed.