Proteomic Analysis of Human Serum by Two-Dimensional Differential Gel Electrophoresis after Depletion of High-Abundant Proteins

Chromy, Brett A.; Gonzales, Arlene D.; Perkins, Julie; Choi, Megan; Corzett, Michele; Chang, Brian C.; Corzett, Christopher H.; McCutchen-Maloney, Sandra L.

doi:10.1021/pr049921p

Cited by 171 publications

(165 citation statements)

References 32 publications

(49 reference statements)

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“…We used a columnbased multiple immunoaffinity system to remove six proteins (albumin, IgG, ␣ 1 -antitrypsin, IgA, transferrin, and haptoglobin) from human CSF. This depletion technique has been shown in a proteomic study on serum to be superior to three other similar depletion methods and resulted in a 76% increase in the number of protein spots detected (30). When loading the same amount of total protein, our CSF study showed a 99% increase in the number of spots detected ( Fig.…”

Section: Within-gel Analyses: Intraindividual Variations-becausesupporting

confidence: 50%

Comparative Proteomic Analysis of Intra- and Interindividual Variation in Human Cerebrospinal Fluid

Malone

Fagan

et al. 2005

Molecular & Cellular Proteomics

114

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Cerebrospinal fluid (CSF) is a potential source of biomarkers for many disorders of the central nervous system, including Alzheimer disease (AD). Prior to comparing CSF samples between individuals to identify patterns of disease-associated proteins, it is important to examine variation within individuals over a short period of time so that one can better interpret potential changes in CSF between individuals as well as changes within a given individual over a longer time span. In this study, we analyzed 12 CSF samples, composed of pairs of samples from six individuals, obtained 2 weeks apart. Multiaffinity depletion, two-dimensional DIGE, and tandem mass spectrometry were used. A number of proteins whose abundance varied between the two time points was identified for each individual. Some of these proteins were commonly identified in multiple individuals. More importantly, despite the intraindividual variations, hierarchical clustering and multidimensional scaling analysis of the proteomic profiles revealed that two CSF samples from the same individual cluster the closest together and that the between-subject variability is much larger than the within-subject variability. Among the six subjects, comparison between the four cognitively normal and the two very mildly demented subjects also yielded some proteins that have been identified in previous AD biomarker studies. These results validate our method of identifying differences in proteomic profiles of CSF samples and have important implications for the design of CSF biomarker studies for AD and other central nervous system disorders. Cerebrospinal fluid (CSF) 1 is produced mainly by the choroid plexus within the ventricles of the brain. It circulates through the ventricular system and around the outside of the brain and spinal cord in the subarachnoid space. CSF is clinically accessible through standard lumbar puncture techniques. Because it is in direct contact with the brain interstitial fluid, biochemical changes taking place in the brain are often reflected in CSF. These features make CSF potentially a very useful source of biomarkers for diagnosis and response to treatment as well as for providing information on pathological processes underlying a number of central nervous system disorders, including Alzheimer disease (AD) (for reviews, see Refs. 1 and 2). Based on known pathology and the hypothesized etiology of AD, a few AD biomarkers in CSF have been identified that may differentiate groups of individuals with clinical disease from those who are cognitively normal, including amyloid ␤ (A␤) 42, total tau, phospho-tau, isoprostane, and sulfatide (3-7). Although some candidate biomarkers have shown promise, each has its limitations. Researchers are still searching for biomarkers that have a higher sensitivity and specificity to differentiate AD from normal aging and other dementias, especially in the early stages. It would also be especially useful to develop antecedent biomarkers for AD, considering its estimated preclinical phase of 10 -20 years (8).Ne...

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Section: Within-gel Analyses: Intraindividual Variations-becausesupporting

confidence: 50%

Comparative Proteomic Analysis of Intra- and Interindividual Variation in Human Cerebrospinal Fluid

Malone

Fagan

et al. 2005

Molecular & Cellular Proteomics

114

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“…1). Recent papers have also confirmed the marked increase in detectable proteins due to immunodepletion [8,9]. Western blot analysis of raw and immunodepleted serum using an antibody to albumin also shows the effectiveness of the immuno-depletion column (Fig.…”

Section: Immunodepletion Of High-abundant Serum Proteinsmentioning

confidence: 64%

2‐D difference gel electrophoresis of the lung squamous cell carcinoma versus normal sera demonstrates consistent alterations in the levels of ten specific proteins

et al. 2007

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Research Article 2-D difference gel electrophoresis of the lung squamous cell carcinoma versus normal sera demonstrates consistent alterations in the levels of ten specific proteinsMost lung cancers are diagnosed too late for curative treatment to be possible, therefore early detection is crucial. Serum proteins are a rich source of biomarkers and have the potential to be used as diagnostic and prognostic indicators for lung cancer. In order to examine differences in serum levels of specific proteins associated with human lung squamous carcinoma, immunodepletion of albumin and five other high-abundant serum proteins followed by 2-D difference gel electrophoresis (DIGE) analysis and subsequent MS was used to generate a panel of proteins found to be differentially expressed between the cancer and normal samples. Proteins found to have increased abundance levels in squamous cell carcinoma sera compared to normal sera included apolipoprotein A-IV precursor, chain F; human complement component C3c, haptoglobin, serum amyloid A protein precursor and Ras-related protein Rab-7b. Proteins found to have lower abundance levels in squamous cell carcinoma sera compared to normal sera included alpha-2-HS glycoprotein, hemopexin precursor, proapolipoprotein, antithrombin III and SP40; 40. The data presented here demonstrate that high-abundant protein removal combined with 2-D DIGE is a powerful strategy for the discovery of potential biomarkers. The identification of lung cancer-specific biomarkers is crucial to early detection, which in turn could lead to a dramatic increase in survival rates. IntroductionOver 1 million people are diagnosed with lung cancer each year [1]. Most lung cancer is diagnosed too late for curative treatment to be possible. Efforts at early detection and treatment have had little success to date and hence the overall prognosis remains poor. In the majority of those diagnosed with lung cancer, the disease has already metastasised at the time of diagnosis. Lung cancer comprises broadly of two groups, small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC). NSCLC comprises more than 80% of lung cancers including squamous cell carcinoma, adenocarcinoma and large cell carcinoma, with squamous carcinoma being the most common type.A major factor in the high mortality of lung cancer patients is the presence of metastatic tumours in approximately two-thirds of patients at time of diagnosis [2]. Detection of cancer in these patients at earlier stages would increase survival rates dramatically. The identification of lung cancer-specific biomarkers is critical to early detection. The pathophysiology of lung squamous cell carcinoma development is complex and incompletely understood. Genetic alterations involved in the pathogenesis of lung cancer produce proteins involved in cell growth, invasion/ metastasis, differentiation, cell cycle processes, apoptosis and angiogenesis. Discovering these mechanisms and pathways will undoubtedly lead to new ways in dealing with prevention, early detection an...

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“…In this study, we build on this approach by coupling multi-component immunoaffinity subtraction with N-glycopeptide enrichment for comprehensive 2-D LC-MS/MS analysis of the human plasma N-glycoproteome. A conservatively estimated dynamic range of detection of >10 8 was achieved by effectively reducing the protein concentration range and overall sample complexity. This overall dynamic range of detection enabled confident identification of 303 non-redundant N-glycoproteins, many of which represented low abundance secreted and extracellular proteins.…”

Section: Introductionmentioning

confidence: 99%

Human Plasma N-Glycoproteome Analysis by Immunoaffinity Subtraction, Hydrazide Chemistry, and Mass Spectrometry

et al. 2005

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The enormous complexity, wide dynamic range of relative protein abundances of interest (over 10 orders of magnitude), and tremendous heterogeneity (due to post-translational modifications, such as glycosylation) of the human blood plasma proteome severely challenge the capabilities of existing analytical methodologies. Here, we describe an approach for broad analysis of human plasma Nglycoproteins using a combination of immunoaffinity subtraction and glycoprotein capture to reduce both the protein concentration range and overall sample complexity. Six high-abundance plasma proteins were simultaneously removed using a pre-packed, immobilized antibody column. N-linked glycoproteins were then captured from the depleted plasma using hydrazide resin and enzymatically digested, and the bound N-linked glycopeptides were released using peptide-N-glycosidase F (PNGase F). Following strong cation exchange (SCX) fractionation, the deglycosylated peptides were analyzed by reversed-phase capillary liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Using stringent criteria, a total of 2053 different N-glycopeptides were confidently identified, covering 303 non-redundant N-glycoproteins. This enrichment strategy significantly improved detection and enabled identification of a number of low-abundance proteins, exemplified by interleukin-1 receptor antagonist protein (~200 pg/mL), cathepsin L (~1 ng/mL), and transforming growth factor beta 1 (~2 ng/mL). A total of 639 N-glycosylation sites were identified, and the overall high accuracy of these glycosylation site assignments as assessed by accurate mass measurement using high resolution liquid chromatography coupled to Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR) is initially demonstrated.

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Proteomic Analysis of Human Serum by Two-Dimensional Differential Gel Electrophoresis after Depletion of High-Abundant Proteins

Cited by 171 publications

References 32 publications

Comparative Proteomic Analysis of Intra- and Interindividual Variation in Human Cerebrospinal Fluid

Comparative Proteomic Analysis of Intra- and Interindividual Variation in Human Cerebrospinal Fluid

2‐D difference gel electrophoresis of the lung squamous cell carcinoma versus normal sera demonstrates consistent alterations in the levels of ten specific proteins

Human Plasma N-Glycoproteome Analysis by Immunoaffinity Subtraction, Hydrazide Chemistry, and Mass Spectrometry

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