Protein glycosylation is one of the most common protein modifications, and the quantitative analysis of glycoproteins has the potential to reveal biological functions and their association with ...disease. However, the high throughput accurate quantification of glycoproteins is technically challenging due to the scarcity of robust assays to detect and quantify glycoproteins. Here we describe the development of multiplexed targeted MS assays to quantify N-linked glycosite-containing peptides in serum using parallel reaction monitoring (PRM). Each assay was characterized by its performance metrics and criteria established by the National Cancer Institute’s Clinical Proteomic Tumor Analysis Consortium (NCI CPTAC) to facilitate the widespread adoption of the assays in studies designed to confidently detect changes in the relative abundance of these analytes. An in-house developed software program, MRMPlus, was used to compute assay performance parameters including specificity, precision, and repeatability. We show that 43 selected N-linked glycosite-containing peptides identified in prostate cancer tissue studies carried out in our group were detected in the sera of prostate cancer patients within the quantitative range of the developed PRM assays. A total of 41 of these formerly N-linked glycosite-containing peptides (corresponding to 37 proteins) were reproducibly quantified based on their relative peak area ratios in human serum during PRM assay development, with 4 proteins showing differential significance in serum from nonaggressive (NAG) vs aggressive (AG) prostate cancer patient serum (n = 50, NAG vs AG). The data demonstrate that the assays can be used for the high throughput and reproducible quantification of a panel of formerly N-linked glycosite-containing peptides. The developed assays can also be used for the quantification of formerly N-linked glycosite-containing peptides in human serum irrespective of disease state.
Mucopolysaccharidosis IVA (MPS IVA) is a rare autosomal recessive lysosomal storage disorder resulting from N-acetylgalactosamine-6-sulfatase (GALNS) deficiency that occurs in approximately 1 in 76 ...000 to 1 in 640 000 live births. Given that the diagnosis of MPS IVA relies heavily on the results of initial urine glycosaminoglycan (GAG) screening, cases that present with falsely normal urine GAG concentrations can delay the diagnosis and follow-up care for patients. This case study follows a patient diagnosed with MPS IVA at 9 months of age based on relation to a consanguineous 3-year-old sibling with MPS IVA and the use of direct enzyme activity analysis. Details regarding skeletal presentation and identification of genetic variants are presented along with data on follow-up urinary GAG monitoring during treatment with enzyme replacement therapy and treatment for a growth hormone disorder.
Clinical proteomics requires large-scale analysis of human specimens to achieve statistical significance. We evaluated the long-term reproducibility of an iTRAQ (isobaric tags for relative and ...absolute quantification)-based quantitative proteomics strategy using one channel for reference across all samples in different iTRAQ sets. A total of 148 liquid chromatography tandem mass spectrometric (LC–MS/MS) analyses were completed, generating six 2D LC–MS/MS data sets for human-in-mouse breast cancer xenograft tissues representative of basal and luminal subtypes. Such large-scale studies require the implementation of robust metrics to assess the contributions of technical and biological variability in the qualitative and quantitative data. Accordingly, we derived a quantification confidence score based on the quality of each peptide-spectrum match to remove quantification outliers from each analysis. After combining confidence score filtering and statistical analysis, reproducible protein identification and quantitative results were achieved from LC–MS/MS data sets collected over a 7-month period. This study provides the first quality assessment on long-term stability and technical considerations for study design of a large-scale clinical proteomics project.
Ovarian cancer is the most lethal gynecologic malignancy in women, and high-grade serous ovarian cancer (HGSOC) is the most common subtype. Currently, no clinical test has been approved by the FDA to ...screen the general population for ovarian cancer. This underscores the critical need for the development of a robust methodology combined with novel technology to detect diagnostic biomarkers for HGSOC in the sera of women. Targeted mass spectrometry (MS) can be used to identify and quantify specific peptides/proteins in complex biological samples with high accuracy, sensitivity, and reproducibility. In this study, we sought to develop and conduct analytical validation of a multiplexed Tier 2 targeted MS parallel reaction monitoring (PRM) assay for the relative quantification of 23 putative ovarian cancer protein biomarkers in sera.
To develop a PRM method for our target peptides in sera, we followed nationally recognized consensus guidelines for validating fit-for-purpose Tier 2 targeted MS assays. The endogenous target peptide concentrations were calculated using the calibration curves in serum for each target peptide. Receiver operating characteristic (ROC) curves were analyzed to evaluate the diagnostic performance of the biomarker candidates.
We describe an effort to develop and analytically validate a multiplexed Tier 2 targeted PRM MS assay to quantify candidate ovarian cancer protein biomarkers in sera. Among the 64 peptides corresponding to 23 proteins in our PRM assay, 24 peptides corresponding to 16 proteins passed the assay validation acceptability criteria. A total of 6 of these peptides from insulin-like growth factor-binding protein 2 (IBP2), sex hormone-binding globulin (SHBG), and TIMP metalloproteinase inhibitor 1 (TIMP1) were quantified in sera from a cohort of 69 patients with early-stage HGSOC, late-stage HGSOC, benign ovarian conditions, and healthy (non-cancer) controls. Confirming the results from previously published studies using orthogonal analytical approaches, IBP2 was identified as a diagnostic biomarker candidate based on its significantly increased abundance in the late-stage HGSOC patient sera compared to the healthy controls and patients with benign ovarian conditions.
A multiplexed targeted PRM MS assay was applied to detect candidate diagnostic biomarkers in HGSOC sera. To evaluate the clinical utility of the IBP2 PRM assay for HGSOC detection, further studies need to be performed using a larger patient cohort.
Accurate protein identification and quantitation are critical when interpreting the biological relevance of large-scale shotgun proteomics data sets. Although significant technical advances in ...peptide and protein identification have been made, accurate quantitation of high-throughput data sets remains a key challenge in mass spectrometry data analysis and is a labor intensive process for many proteomics laboratories. Here, we report a new SILAC-based proteomics quantitation software tool, named IsoQuant, which is used to process high mass accuracy mass spectrometry data. IsoQuant offers a convenient quantitation framework to calculate peptide/protein relative abundance ratios. At the same time, it also includes a visualization platform that permits users to validate the quality of SILAC peptide and protein ratios. The program is written in the C# programming language under the Microsoft .NET framework version 4.0 and has been tested to be compatible with both 32-bit and 64-bit Windows 7. It is freely available to noncommercial users at http://www.proteomeumb.org/MZw.html.
The major initiative of this study was to implement a novel proteomic approach in order to detect protein carbonylation in aged mouse brain. Several lines of evidence indicate that reactive oxygen ...species (ROS)-induced protein oxidation plays an essential role in the initiation of age-related neuropathologies. Therefore, the identification of free radical or peroxide substrates would provide further insight into key biochemical mechanisms that contribute to the progression of certain neurological disorders.
Historically, ROS targets have been identified by conventional immunological two-dimensional (2-D) gel electrophoresis and mass spectrometric analyses. However, specific classes of proteins, such as transmembrane-spanning proteins, high-molecular-weight proteins, and very acidic or basic proteins, are frequently excluded or underrepresented by these analyses. In order to fill this technologic gap, we have used a functional proteomics approach using a liquid chromatography tandem mass spectrometric (LC-MS/MS) analysis coupled with a hydrazide biotin-streptavidin methodology in order to identify protein carbonylation in aged mice.
Our initial studies suggest an ability to identify at least 100 carbonylated proteins in a single LC-MS/MS experiment. In addition to high-abundance cytosolic proteins that have been previously identified by 2-D gel electrophoresis and mass spectrometric analyses, we are able to identify several low-abundance receptor proteins, mitochondrial proteins involved in glucose and energy metabolism, as well as a series of receptors and tyrosine phosphatases known to be associated with insulin and insulin-like growth factor metabolism and cell-signaling pathways.
Here we describe a rapid and sensitive proteomic analysis for the identification of carbonylated proteins in mouse brain homogenates through the conjunction of liquid chromatography and tandem mass spectrometry methods. We believe the ability to detect these post-translationally modified proteins specifically associated with brain impairments during the course of aging should allow one to more closely and objectively monitor the efficacy of various clinical treatments. In addition, the discovery of these unique brain biomarkers could also provide a conceptual framework for the future design of alternative drugs in the treatment of a variety of age-related neurodegenerative disorders.
Radiation and drug resistance are significant challenges in the treatment of locally advanced, recurrent and metastatic breast cancer that contribute to mortality. Clinically, radiotherapy requires ...oxygen to generate cytotoxic free radicals that cause DNA damage and allow that damage to become fixed in the genome rather than repaired. However, approximately 40% of all breast cancers have hypoxic tumor microenvironments that render cancer cells significantly more resistant to irradiation. Hypoxic stimuli trigger changes in the cell death/survival pathway that lead to increased cellular radiation resistance. As a result, the development of noninvasive strategies to assess tumor hypoxia in breast cancer has recently received considerable attention. Exosomes are secreted nanovesicles that have roles in paracrine signaling during breast tumor progression, including tumor-stromal interactions, activation of proliferative pathways and immunosuppression. The recent development of protocols to isolate and purify exosomes, as well as advances in mass spectrometry-based proteomics have facilitated the comprehensive analysis of exosome content and function. Using these tools, studies have demonstrated that the proteome profiles of tumor-derived exosomes are indicative of the oxygenation status of patient tumors. They have also demonstrated that exosome signaling pathways are potentially targetable drivers of hypoxia-dependent intercellular signaling during tumorigenesis. This article provides an overview of how proteomic tools can be effectively used to characterize exosomes and elucidate fundamental signaling pathways and survival mechanisms underlying hypoxia-mediated radiation resistance in breast cancer.
Post-translational modification by ubiquitin is a fundamental regulatory mechanism that is implicated in many cellular processes including the cell cycle, apoptosis, cell adhesion, angiogenesis, and ...tumor growth. The low stoichiometry of ubiquitylation presents an analytical challenge for the detection of endogenously modified proteins in the absence of enrichment strategies. The recent availability of antibodies recognizing peptides with Lys residues containing a di-Gly ubiquitin remnant (K-ε-GG) has greatly improved the ability to enrich and identify ubiquitylation sites from complex protein lysates via mass spectrometry. To date, there have not been any published studies that quantitatively assess the changes in endogenous ubiquitin-modification protein stoichiometry status at the proteome level from different tissues.
In this study, we applied an integrated quantitative mass spectrometry based approach using isobaric tags for relative and absolute quantitation (iTRAQ) to interrogate the ubiquitin-modified proteome and the cognate global proteome levels from luminal and basal breast cancer patient-derived xenograft tissues. Among the proteins with quantitative global and ubiquitylation data, 91 % had unchanged levels of total protein relative abundance, and less than 5 % of these proteins had up- or down-regulated ubiquitylation levels. Of particular note, greater than half of the proteins with observed changes in their total protein level also had up- or down-regulated changes in their ubiquitylation level.
This is the first report of the application of iTRAQ-based quantification to the integrated analysis of the ubiquitylated and global proteomes at the tissue level. Our results underscore the importance of conducting integrated analyses of the global and ubiquitylated proteomes toward elucidating the specific functional significance of ubiquitylation.
Alterations in the trafficking and function of the endocytic pathway have been extensively documented to be one of the earliest pathological changes in sporadic Alzheimer's disease (AD). Although the ...pathophysiological consequences of these endosomal/lysosomal changes are currently unknown, several recent studies have suggested that such changes in endocytic function are able to cause a redistribution of several lysosomal hydrolases into early endosomes, leading to the overproduction of neurotoxic amyloid peptide. Recently, we and others have demonstrated that abnormal endocytic pathology within post-mitotic neurons can, in part, be attributed to alterations in sphingomyelin/ceramide metabolism, resulting in the intracellular accumulation of ceramide. Once inside the cell, the ability of ceramide to physically alter membrane structure, formation, and fusion, rather than serving solely as a lipid secondary messenger, may severely compromise normal endocytic trafficking. In this review, we will discuss the potential pathological effects of abnormal sphingomyelin/ceramide metabolism on intracellular vesicular transport in relation to both amyloid accumulation in AD and various neurodegenerative diseases associated with lysosomal abnormalities.
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