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1.  Quantitative proteomics in cardiovascular research: global and targeted strategies 
Extensive technical advances in the past decade have substantially expanded quantitative proteomics in cardiovascular research. This has great promise for elucidating the mechanisms of cardiovascular diseases (CVD) and the discovery of cardiac biomarkers used for diagnosis and treatment evaluation. Global and targeted proteomics are the two major avenues of quantitative proteomics. While global approaches enable unbiased discovery of altered proteins via relative quantification at the proteome level, targeted techniques provide higher sensitivity and accuracy, and are capable of multiplexed absolute quantification in numerous clinical/biological samples. While promising, technical challenges need to be overcome to enable full utilization of these techniques in cardiovascular medicine. Here we discuss recent advances in quantitative proteomics and summarize applications in cardiovascular research with an emphasis on biomarker discovery and elucidating molecular mechanisms of disease. We propose the integration of global and targeted strategies as a high-throughput pipeline for cardiovascular proteomics. Targeted approaches enable rapid, extensive validation of biomarker candidates discovered by global proteomics. These approaches provide a promising alternative to immunoassays and other low-throughput means currently used for limited validation.
PMCID: PMC4159306  PMID: 24920501
Cardiovascular diseases; Proteomics; LC-MS; Biomarker; mechanism study; targeted quantification
2.  Surviving the infarct: A profile of cardiac myosin binding protein-C pathogenicity, diagnostic utility, and proteomics in the ischemic myocardium 
Cardiac myosin binding protein-C (cMyBP-C) is a regulatory protein of the contractile apparatus within the cardiac sarcomere. Ischemic injury to the heart during myocardial infarction (MI) results in the cleavage of cMyBP-C in a phosphorylation-dependent manner and release of an N-terminal fragment (C0C1f) into the circulation. C0C1f has been shown to be pathogenic within cardiac tissue, leading to the development of heart failure (HF). Based on its high levels and early release into the circulation post-MI, C0C1f may serve as a novel biomarker for diagnosing MI more effectively than current clinically used biomarkers. Over time, circulating C0C1f could trigger an autoimmune response leading to myocarditis and progressive cardiac dysfunction. Given the importance of cMyBP-C phosphorylation state in the context of proteolytic cleavage and release into the circulation post-MI, understanding the post-translational modifications (PTMs) of cMyBP-C would help in further elucidating the role of this protein in health and disease. Accordingly, recent studies have implemented the latest proteomics approaches to define the PTMs of cMyBP-C. The use of such proteomics assays may provide accurate quantitation of the levels of cMyBP-C in the circulation following MI, which could, in turn, demonstrate the efficacy of using plasma cMyBP-C as a cardiac-specific early biomarker of MI. In this review, we define the pathogenic and potential immunogenic effects of C0C1f on cardiac function in the post-MI heart. We also discuss the most advanced proteomics approaches now used to determine cMyBP-C PTMs with the aim of validating C0C1f as an early biomarker of MI.
PMCID: PMC4162529  PMID: 24888514
Autoantibodies; Biomarkers; cMyBP-C; Dilated cardiomyopathy; Myocardial infarction; Myocarditis; Post-translational modification; Proteomics
3.  Cardiac extracellular proteome profiling and membrane topology analysis using glycoproteomics 
Extracellular proteins are easily accessible, which presents a sub-proteome of molecular targets that have high diagnostic and therapeutic potential. Efforts have been made to catalogue the cardiac extracellular matridome and analyze the topology of identified proteins for the design of therapeutic targets. Although many bioinformatics tools have been developed to predict protein topology, topology has been experimentally validated for only a very small portion of membrane proteins. The aim of this study was to use a glycoproteomics and mass spectrometry approach to identify glycoproteins in the extracellular matridome of the infarcted LV and provide experimental evidence for topological determination.
Experimental design
Glycoproteomics analysis was performed on eight biological replicates of day 7 post-MI samples from wild type mice using solid-phase extraction of glycopeptides, followed by mass spectrometric identification of N-linked glycosylation sites for topology assessment.
We identified hundreds of glycoproteins and the identified N-glycosylation sites provide novel information on the correct topology for membrane proteins present in the infarct setting.
Conclusions and clinical relevance
Our data provides the foundation for future studies of the LV infarct extracellular matridome, which may facilitate the discovery of drug targets and biomarkers.
PMCID: PMC4212811  PMID: 24920555
Extracellular matridome; glycoprotein; membrane orientation; matrix metalloproteinase; proteomics; myocardial infarction; left ventricle
4.  Identification of Cardiac Myofilament Protein Isoforms Using Multiple Mass Spectrometry Based Approaches 
The identification of protein isoforms in complex biological samples is challenging. We, therefore, used a mass spectrometry (MS) approach to unambiguously identify cardiac myofilament protein isoforms based on the observation of a tryptic peptide consisting of a sequence unique to a particular isoform.
Experimental design
Three different workflows were used to isolate and fractionate rat cardiac myofilament subproteomes. All fractions were analyzed on an LTQ-Orbitrap MS, proteins were identified using various search engines (Mascot, X!Tandem, X!Tandem Kscore and OMSSA) with results combined via PepArML Meta-Search Engine, and a post-search analysis was performed by MASPECTRAS.
The combination of multiple workflows and search engines resulted in a larger number of non-redundant proteins identified than with individual methods. A total of 102 myofilament annotated proteins were observed overlapping in two or three of the workflows. Literature search for myofilament presence with manual validation of the MS spectra was carried out for unambiguous identification: 10 cardiac myofilament and 17 cardiac myofilament-associated proteins were identified with 39 isoforms and sub-isoforms.
Conclusion and clinical relevance
We have identified multiple isoforms of myofilament proteins that are present in cardiac tissue using unique tryptic peptides. Changes in distribution of these protein isoforms under pathological conditions could ultimately allow for clinical diagnostics or as therapeutic targets.
PMCID: PMC4222538  PMID: 24974818
5.  Heavy Hitting: Using Water to Label Humans 
Monitoring protein dynamics, compared to measuring static protein expression profiles taken with snapshot evaluations, have recently been the focus of proteomics studies examining tissue or blood samples where time course changes occur. Using deuterium oxide (2H2O) to label amino acids is a useful method to monitor protein turnover rates. The synthesis rate for individual proteins is calculated from the rate of 2H incorporation into specific proteins analyzed by high resolution mass spectrometry. In this issue, Wang and colleagues measured the plasma protein turnover dynamics in healthy humans by in vivo 2H2O labeling [Wang et al Proteomics 2014]. The authors developed and validated a safe and accessible 2H2O administration protocol to record the turnover dynamics of 542 plasma proteins using mass spectrometry. Their study demonstrates a promising new way to evaluate plasma protein dynamics in clinical trials where such knowledge could help for prognosis and evaluating treatment efficacy.
PMCID: PMC4230989  PMID: 25044642
deuterium oxide; mass spectrometry; matrix metalloproteinase; protein turnover; proteomics
6.  Top-down Mass Spectrometry of Cardiac Myofilament Proteins in Health and Disease 
Myofilaments are composed of thin and thick filaments which coordinate with each other to regulate muscle contraction and relaxation. Posttranslational modifications (PTMs) together with genetic variations and alternative splicing of the myofilament proteins play essential roles in regulating cardiac contractility in health and disease. Therefore, a comprehensive characterization of the myofilament proteins in physiological and pathological conditions is essential for better understanding the molecular basis of cardiac function and dysfunction. Due to the vast complexity and dynamic nature of proteins, it is challenging to obtain a holistic view of myofilament protein modifications. In recent years, top-down mass spectrometry (MS) has emerged as a powerful approach to study isoform composition and PTMs of proteins owing to its advantage of complete sequence coverage and its ability to identify PTMs and sequence variants without a priori knowledge. In this review, we will discuss the application of top-down MS to study cardiac myofilaments and highlight the insights it provides into the understanding of molecular mechanisms in contractile dysfunction of heart failure. Particularly, recent results of cardiac troponin and tropomyosin modifications will be elaborated. The limitations and perspectives on the use of top-down MS for myofilament protein characterization will also be briefly discussed.
PMCID: PMC4231170  PMID: 24945106
7.  Epigenomes: the missing heritability in human cardiovascular disease? 
Cardiovascular disease is a tremendous burden on human health and results from malfunction of various networks of biological molecules in the context of environmental stress. Despite strong evidence of heritability, many common forms of heart disease (heart failure in particular) have not yielded to genome-wide association studies to identify causative mutations acting via the disruption of individual molecules. Increasing evidence suggests, however, that genetic variation in non-coding regions is strongly linked to disease susceptibility. We hypothesize that epigenomic variation may engender different chromatin environments in the absence of (or in parallel with) changes in protein or mRNA sequence and abundance. In this manner, distinct—genetically encoded—chromatin environments can exhibit distinct responses to environmental stresses that cause heart failure, explaining a significant portion of the altered susceptibility that is observed in human disease.
PMCID: PMC4267468  PMID: 24957631
chromatin; genomics; heart failure; human studies; systems biology
8.  A Method for Isolation and Identification of Urinary Biomarkers in Patients with Diabetic Nephropathy 
The poor performance of current tests for predicting the onset, progression and treatment response of diabetic nephropathy has engendered a search for more sensitive and specific urinary biomarkers. Our goal was to develop a new method for protein biomarker discovery in urine from these patients.
Experimental Design
We analyzed urine from normal subjects and patients with early and advanced nephropathy. Proteins were separated using a novel analysis process including immunodepletion of high abundance proteins followed by two stage LC fractionation of low abundance proteins. The proteins in the fractions were sequenced using MS/MS.
Immunodepletion of selected high abundance proteins followed by two stage LC produced approximately 700 fractions, each less complex and more amenable to analysis than the mixture and requiring minimal processing for MS identification. Comparison of fractions between normal and diabetic nephropathy subjects revealed several low abundance proteins that reproducibly distinguished low glomerular filtration rate (GFR) from both high GFR diabetic and normal subjects, including uteroglobin, a protein previously associated with renal scarring.
Conclusions and clinical relevance
We developed a novel method to identify low abundance urinary proteins that enables the discovery of potential biomarkers to improve the diagnosis and management of patients with diabetic nephropathy.
PMCID: PMC4517596  PMID: 21956890
diabetic nephropathy; immunodepletion; liquid chromatography; proteomic methods; urinary biomarkers
9.  Improved Signal Processing and Normalization for Biomarker Protein Detection in Broad-Mass-Range TOF Mass Spectra from Clinical Samples 
To demonstrate robust detection of biomarkers in broad-mass-range TOF-MS data.
Experimental Design
Spectra were obtained for two serum protein profiling studies: (1) 2–200 kDa for 132 patients, 67 healthy and 65 diagnosed as having adult T-cell leukemia and (2) 2–100 kDa for 140 patients, 70 pairs, each with matched prostate-specific-antigen (PSA) levels and biopsy-confirmed diagnoses of one benign and one prostate cancer. Signal processing was performed on raw spectra and peak data were normalized using four methods. Feature selection was performed using Bayesian network analysis and a classifier was tested on withheld data. Identification of candidate biomarkers was pursued.
Integrated peak intensities were resolved over full spectra. Normalization using local noise values was superior to global methods in reducing peak correlations, reducing replicate variability, and improving feature selection stability. For the leukemia data set, potential disease biomarkers were detected and were found to be predictive for withheld data. Preliminary assignments of protein IDs were consistent with published results and LC-MS/MS identification. No PSA-independent biomarkers were detected in the prostate cancer data set.
Conclusions and clinical relevance
Signal processing, local SNR normalization and BNA feature selection facilitate robust detection and identification of biomarker proteins in broad-mass-range clinical TOF-MS data.
PMCID: PMC4503319  PMID: 21751409
normalization; protein profiling; serum biomarkers; signal processing; TOF-MS
10.  Carboxypeptidases in disease: Insights from peptidomic studies 
Carboxypeptidases (CPs) perform many diverse physiological functions by removing C-terminal amino acids from proteins and peptides. Some CPs function in the degradation of proteins in the digestive tract while other enzymes play biosynthetic roles in the formation of neuropeptides and peptide hormones. Another set of CPs modify tubulin by removing amino acids from the C-terminus and from polyglutamyl side chains, thereby altering the properties of microtubules. This review focuses on three CPs: carboxypeptidase E, carboxypeptidase A6, and cytosolic carboxypeptidase 1. Naturally-occurring mutations in all three of these enzymes are associated with disease phenotypes, ranging from obesity to epilepsy to neurodegeneration. Peptidomics is a useful tool to investigate the relationship between these mutations and alterations in peptide levels. This technique has also been used to define the function and characteristics of CPs. Results from peptidomics studies have helped to elucidate the function of CPs and clarify the biological underpinnings of pathologies by identifying peptides altered in disease states. This review describes the use of peptidomic techniques to gain insights into the normal function of CPs and the molecular defects caused by mutations in the enzymes.
PMCID: PMC4062080  PMID: 24470285
11.  Aminopeptidase Activities as Prospective Urinary Biomarkers for Bladder Cancer 
Proteases have been implicated in cancer progression and invasiveness. We have investigated the activities, as opposed to simple protein levels, of selected aminopeptidases in urine specimens to serve as potential novel biomarkers for urothelial cancer.
Experimental design
The unique urinary proteomes of males and females were profiled to establish the presence of a gender-independent set of aminopeptidases. Samples were also collected from patients with urothelial cancer and matched controls. A SOP for urine processing was developed taking into account hydration variation. Five specific aminopeptidase activity assays, using fluorophoric substrates, were optimized for evaluation of marker potential.
Nineteen exopeptidases and 21 other proteases were identified in urine and the top-5 most abundant aminopeptidases, identical in both genders, selected for functional studies. Depending on the enzyme, activities were consistently lower (P ≤ 0.05), higher or unchanged in the cancer samples as compared to controls. Two selected aminopeptidase activities used as a binary classifier resulted in a ROC curve with an AUC = 0.898.
Conclusion and clinical relevance
We have developed functional assays that characterize aminopeptidase activities in urine specimens with adequate technical and intra-individual reproducibility. With further testing, it could yield a reliable biomarker test for bladder cancer detection or prognostication.
PMCID: PMC4059539  PMID: 24591208
activity biomarkers; aminopeptidases; bladder cancer; fluorescence; urine
12.  Cathepsin B: Multiple roles in cancer 
Proteases, including intracellular proteases, play roles at many different stages of malignant progression. Our focus here is cathepsin B, a lysosomal cysteine cathepsin. High levels of cathepsin B are found in a wide variety of human cancers, levels that often induce secretion and association of cathepsin B with the tumor cell membrane. In experimental models, such as transgenic models of murine pancreatic and mammary carcinomas, causal roles for cathepsin B have been demonstrated in initiation, growth/tumor cell proliferation, angiogenesis, invasion, and metastasis. Tumor growth in transgenic models is promoted by cathepsin B in tumor-associated cells, for example, tumor-associated macrophages, as well as in tumor cells. In transgenic models, the absence of cathepsin B has been associated with enhanced apoptosis, yet cathepsin B also has been shown to contribute to apoptosis. Cathepsin B is part of a proteolytic pathway identified in xenograft models of human glioma; targeting only cathepsin B in these tumors is less effective than targeting cathepsin B in combination with other proteases or protease receptors. Understanding the mechanisms responsible for increased expression of cathepsin B in tumors and association of cathepsin B with tumor cell membranes is needed to determine whether targeting cathepsin B could be of therapeutic benefit.
PMCID: PMC4205946  PMID: 24677670
Cancer; Cathepsin B; Cysteine proteases
13.  Exploration of the normal human bronchoalveolar lavage fluid proteome 
We obtained insight into normal lung function by proteome analysis of bronchoalveolar lavage fluid (BALF) from six normal human subjects using a “Lyse-N-Go’ shotgun proteomic protocol. Intra-sample variation was calculated using three different label-free methods, (i) protein sequence coverage; (ii) peptide spectral counts and (iii) peptide single-ion current areas (PICA), which generates protein expression data by summation of the area under the curve for a given peptide single-ion current trace and then adding values for all peptides from that same parent protein. PICA gave the least intra-subject variability and was used to calculate differences in protein expression between the six subjects. We observed an average threefold inter-sample variability, which affects analysis of changes in protein expression that occur in different diseases. We detected 167 unique proteins with >100 proteins detected in each of the six individual BAL samples, 42 of which were common to all six subjects. Gene ontology analysis demonstrated enrichment of several biological processes in the lung, reflecting its expected role in gas exchange and host defense as an immune organ. The same biological processes were enriched compared to either plasma or total genome proteome, suggesting an active enrichment of plasma proteins in the lung rather than passive capillary leak.
PMCID: PMC4432467  PMID: 21136857
Bronchoalveolar lavage; Lung; Normal
14.  Can proteomics yield insight into aging aorta? 
The aging aorta exhibits structural and physiological changes that are reflected in the proteome of its component cells types. The advance in proteomic technologies has made it possible to analyze the quantity of proteins associated with the natural history of aortic aging. These alterations reflect the molecular and cellular mechanisms of aging and could provide an opportunity to predict vascular health. This paper focuses on whether discoveries stemming from the application of proteomic approaches of the intact aging aorta or vascular smooth muscle cells can provide useful insights. Although there have been limited studies to date, a number of interesting proteins have been identified that are closely associated with aging in the rat aorta. Such proteins, including milk fat globule-EGF factor 8(MFG-E8), matrix metalloproteinase type-2 (MMP2), and vitronectin, could be used as indicators of vascular health, or even explored as therapeutic targets for aging-related vascular diseases.
PMCID: PMC4429130  PMID: 23788441
proteomics; aging aorta; biomarker; therapeutic target; MFG-E8
15.  Translational Vitreous Proteomics 
Proteomic analysis is important in the examination of complex extracellular matrices such as the vitreous where several tissues both inside the eye and remote to the eye contribute to the diseased state. In these cases, genomic analysis of local tissue gene expression may be insufficient or misleading. By switching the emphasis from diagnostic biomarkers to biomarkers with therapeutic potential, we can create rationale therapeutic strategies for blinding vitreoretinal diseases. The same strategy can extend to other ocular tissues. We are just beginning to understand the molecular constituents of the vitreous in health and disease, and translational proteomics may more effectively direct efforts to cure blindness.
PMCID: PMC3964148  PMID: 24115652
16.  A Biorepository for Ophthalmic Surgical Specimens 
Biorepositories are collections of surgically obtained human tissues for current and future investigations of disease mechanisms, therapeutics, and diagnostics. In ophthalmology, a critical challenge is how to interface the operating room with the laboratory. To attain standards required for basic research, clinical and research teams must cooperate to collect, annotate, and store specimens that yield consistent results required for advanced molecular techniques. We developed an efficient platform for obtaining vitreous and other eye tissues from the operating room and transferring them to the lab. The platform includes a mobile lab cart for on-site tissue processing, a multi-user, web-based database for point-of-care phenotypic capture, and an integrated data tracking system for long-term storage. These biorepository instruments have proven essential for our studies in ophthalmic disease proteomics. This system can be implemented in other operating rooms and laboratories for a variety of biological tissues.
PMCID: PMC3964151  PMID: 24115637
17.  A Decade of the Proteomics Studies of Glaucomatous Neurodegeneration 
Glaucoma is a leading cause of blindness; however, limited understanding of the molecular mechanisms involved in optic nerve degeneration hinders the development of improved treatment strategies. Proteomics techniques that combine the protein chemistry, mass spectrometry, and bioinformatics offer the opportunity to shed light on molecular mechanisms so that new treatment strategies can be developed for immunomodulation, neuroprotection, neurorescue, neuroregeneration, and function gain in glaucoma. The proteomics technologies also hold great promise for biomarker discovery, another important goal of glaucoma research. As much as developing new treatment strategies, molecular biomarkers are strongly needed for early diagnosis of glaucoma, prediction of its prognosis, and monitoring the responses to new treatments. It is now a decade that the proteomics analysis techniques have been using to move glaucoma research forward. This review will focus on valuable applications of proteomics in the field of glaucoma research and highlight the power of this analytical toolbox in translational and clinical research towards better characterization and improved treatment of glaucomatous neurodegeneration and discovery of glaucoma-related molecular biomarkers.
PMCID: PMC4011134  PMID: 24415558
Biomarker; Glaucoma; Neurodegeneration; Proteomics
18.  Aberrant Glycosylation in the Human Trabecular Meshwork 
To determine the difference in protein glycosylation and glycosylation enzyme levels between glaucomatous and control trabecular meshwork (TM).
Experimental design
Glaucomatous and normal donor (n=12 each) TM tissues, Lectin-fluorescence, fluorophore assisted carbohydrate analyses, and quantitative mass spectrometry were used to determine the glycosylation levels. Primary TM cells and glycosylation inhibitors were used to determine their effect on cell shape and motility.
In contrast to elevated levels of glycoproteins determined by lectin-fluorescence, simultaneous hyper and hypo-glycosylation in glaucomatous trabecular meshwork was revealed by fluorophore assisted carbohydrate analyses. Analyses of enzymes showed elevation of Beta-glycosidase 1 and decrease in Galactosyltransferase family 6 domain containing protein 1 in the glaucomatous trabecular meshwork. Quantitative mass spectrometry identified select protein level changes between glaucomatous and normal trabecular meshwork. Primary trabecular meshwork cells were treated with inhibitors to elicit hypo-glycosylation, which affected cell shape, motility, and fluorescent tracer transport across a layer of trabecular meshwork cells.
Conclusions and clinical relevance
Global protein glycosylation is aberrant in glaucomatous trabecular meshwork compared to controls. The results presented here suggest that the alteration in global TM protein glycosylation encompassing cellular and extracellular matrix proteins contributes to glaucoma pathology likely mediated through changes in properties of TM cells.
PMCID: PMC4015974  PMID: 24458570
Trabecular meshwork; glycosylation; carbohydrate electrophoresis; glaucoma
19.  The Role of O-GlcNAc Signaling in the Pathogenesis of Diabetic Retinopathy 
Diabetic retinopathy is a leading cause of blindness worldwide. Despite laser and surgical treatments, anti-angiogenic and other therapies, and strict metabolic control, many patients progress to visual impairment and blindness. New insights are needed into the pathophysiology of diabetic retinopathy in order to develop new methods to improve the detection and treatment of disease and the prevention of blindness. Hyperglycemia and diabetes result in increased flux through the hexosamine biosynthetic pathway, which, in turn, results in increased post-translational modification of Ser/Thr residues of proteins by O-linked β-N-acetylglucosamine (O-GlcNAc). O-GlcNAcylation is involved in regulation of many nuclear and cytoplasmic proteins in a manner similar to protein phosphorylation. Altered O-GlcNAc signaling has been implicated in the pathogenesis of diabetes and may play an important role in the pathogenesis of diabetic retinopathy. The goal of this review is to summarize the biology of the hexosamine biosynthesis pathway and O-GlcNAc signaling, to present the current evidence for the role of OGlcNAc signaling in diabetes and diabetic retinopathy, and to discuss future directions for research on O-GlcNAc in the pathogenesis of diabetic retinopathy.
PMCID: PMC4037871  PMID: 24550151
diabetes; diabetic retinopathy; glucose toxicity; hexosamine biosynthesis pathway; O-GlcNAcylation
20.  Proteomics and the Eye 
PMCID: PMC4346283  PMID: 24729286
21.  Robust microarray production of freshly expressed proteins in a human milieu 
In vitro transcription/translation (IVTT) systems are widely used in proteomics. For clinical applications, mammalian systems are preferred for protein folding and activity; however, the level of protein obtained is low. A new system extracted from human cells (1-Step Human Coupled IVT) has the potential to overcome this problem and deliver high yields of protein expressed in a human milieu.
Experimental design
Western blots and self-assembled protein microarrays were used to test the efficiency of protein synthesis by 1-Step Human Coupled IVT (HCIVT) compared to rabbit reticulocyte lysate (RRL). The arrays were also used to measure the immune response obtained from serum of patients exposed to pathogens or vaccine.
HCIVT performed better than RRL in all experiments. The yield of protein synthesized in HCIVT is more than 10 times higher than RRL, in both western blot and protein microarrays. Moreover, HCIVT showed a robust lot-to-lot reproducibility. In immune assays, the signals of many antigens were detected only in HCIVT-expressed arrays, mainly due to the reduction in the background signal and the increased levels of protein on the array.
Conclusion and clinical relevance
HCIVT is a robust IVTT system that yields high levels of protein produced in a human milieu. It can be used in applications where protein expression in a mammalian system and high yields are needed. The increased immunogenic response of HCIVT-expressed proteins will be critical for biomarker discovery in many diseases, including cancer.
PMCID: PMC4369765  PMID: 23027544
NAPPA; protein microarray; in vitro transcription and translation; Human Coupled IVT
22.  Hemoglobin Depletion from plasma: Considerations for Proteomic Discovery in Sickle Cell Disease and other Hemolytic Processes 
Proteomics. Clinical applications  2010;4(12):926-930.
Hemoglobin (Hb) depletion with nickel affinity chromatography has been shown to increase the number of proteins identified in proteomic studies of erythrocytes, but limited data exist on the application of this technique in depletion of Hb from plasma or serum required for clinical biomarker studies. The aim of this study was to explore the potential of using nickel-beads for Hb depletion of plasma.
Experimental design
Nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography was used to deplete Hb from hemolyzed plasma samples obtained from children with sickle cell disease (SCD, n=7) and normal human plasma (n=4). Ni-NTA bound proteins were analyzed by one-dimensional gel electrophoresis, followed by in-gel digestion for characterization using a LTQ-Orbitrap hybrid mass spectrometer. In addition, the loss of two non-hemoglobin related plasma proteins, thrombospondin1 (TSP1) and L-selectin, by Ni-NTA was determined by ELISA (SCD n=6, non-SCD controls n=2).
Ni-NTA resulted in an average 60% decrease in plasma protein concentration, which was not hemolysis dependent. Specifically, Hb (7 peptides) and the top three proteins, alpha-2-macroglobulin (75 peptides), apolipoprotein B-100 (73 peptides), and albumin (42 peptides) were Ni-NTA bound. In addition, using an ELISA assay two non-hemoglobin associated plasma proteins TSP1 and L-selectin were decreased by Ni-NTA.
Conclusions and clinical relevance
Hb depletion with Ni-NTA is effective for Hb removal but is not specific. There is potential for deleterious depletion of potential biomarkers that may limit the applicability of this method. Consideration of alternate methods of Hb depletion for clinical proteomics may be warranted.
PMCID: PMC4364410  PMID: 21179892
Biomarkers; Hemoglobin depletion; Plasma proteomics; Sickle cell disease
23.  Opportunities and Challenges of Proteomics in Pediatric Patients: Circulating Biomarkers After Hematopoietic Stem Cell Transplantation As a Successful Example 
Biomarkers have the potential to improve diagnosis and prognosis, facilitate targeted treatment, and reduce health care costs. Thus, there is great hope that biomarkers will be integrated in all clinical decisions in the near future. A decade ago, the biomarker field was launched with great enthusiasm because mass spectrometry revealed that blood contains a rich library of candidate biomarkers. However, biomarker research has not yet delivered on its promise due to several limitations: (i) improper sample handling and tracking as well as limited sample availability in the pediatric population, (ii) omission of appropriate controls in original study designs, (iii) lability and low abundance of interesting biomarkers in blood, and (iv) the inability to mechanistically tie biomarker presence to disease biology. These limitations as well as successful strategies to overcome them are discussed in this review. Several advances in biomarker discovery and validation have been made in hematopoietic stem cell transplantation, the current most effective tumor immunotherapy, and these could serve as examples for other conditions. This review provides fresh optimism that biomarkers clinically relevant in pediatrics are closer to being realized based on: (i) a uniform protocol for low-volume blood collection and preservation, (ii) inclusion of well-controlled independent cohorts, (iii) novel technologies and instrumentation with low analytical sensitivity, and (iv) integrated animal models for exploring potential biomarkers and targeted therapies.
PMCID: PMC4361082  PMID: 25196024
Biomarkers; Risk-stratification; Proteomics; Pediatrics; Graft-versus-Host Disease (GVHD); Hematopoietic stem cell transplantation (HSCT)
24.  Improved Protein Extractionand Protein Identification from Archival Formalin-fixed Paraffin-embedded Human Aortas 
Evaluate combination of heat and elevated pressure to enhance protein extraction and quality of formaldehyde-fixed (FF), and FF paraffin-embedded (FFPE) aorta for proteomics.
Experiment design
Proteins were extracted from fresh frozen aorta at RT. FF and FFPE aortas (3 months and 15 years) were extracted at RT, heat alone, or a combination of heat and high pressure. Protein yields were compared, and digested peptides from the extracts were analyzed with mass spectrometry.
Combined heat and elevated pressure increased protein yield from human FF or FFPE aorta compared to matched tissues with heat alone (1.5 fold) or at RT (8.3 fold), resulting in more proteins identified and with more sequence coverage. The length of storage did adversely affect the quality of proteins from FF tissue. For long term storage, aorta was preserved better with FFPE than FF alone. Periostin and MGF-E8 were demonstrated suitable for MRM assays from FFPE aorta.
Conclusions and clinical relevance
Combination of heat and high pressure is an effective method to extract proteins from FFPE aorta for downstream proteomics. This method opens the possibility for use of archival and often rare FFPE aortas and possibly other tissues available to proteomics for biomarker discovery and quantification.
PMCID: PMC4340701  PMID: 23339088
aorta; FFPE; formalin-fixed paraffin-embedded; heat and high-pressure protein extraction; mass spectrometry
25.  Prediction of Glycan Motifs Using Quantitative Analysis of Multi-lectin Binding: Motifs on MUC1 Produced by Cultured Pancreatic Cancer Cells 
Lectins are valuable tools for detecting specific glycans in biological samples, but the interpretation of the measurements can be ambiguous due to the complexities of lectin specificities. Here we present an approach to improve the accuracy of interpretation by converting lectin measurements into quantitative predictions of the presence of various glycan motifs. The conversion relies on a database of analyzed glycan array data that provides information on the specificities of the lectins for each of the motifs. We tested the method using measurements of lectin binding to glycans on glycan arrays and found that the combined measurements from several lectins are more accurate than individual measurements for predicting the presence or absence of motifs. We then applied the method to predicting motifs on the protein MUC1 expressed in eight different pancreatic cancer cell lines. Each cell line expressed a unique pattern of MUC1 glycoforms, and the glycoforms significantly differed between MUC1 collected from conditioned media and MUC1 collected from cell lysates. This new method could provide more accurate analyses of glycans in biological sample and make the use of lectins more practical and effective for a broad range of researchers.
PMCID: PMC3929537  PMID: 23956151

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