Glycans play significant roles in physiological and pathological processes. Therefore, quantitative analysis of glycans from normal and disease specimens can provide insight into disease onset and progression. Relative glycan quantification usually requires modification of the glycans with either chromogenic or fluorogenic tags for optical measurement or isotopic tags for mass spectrometric analysis. Due to rapid advances in mass spectrometry (MS) instruments in resolution, sensitivity and speed, MS-based methods have become increasingly popular for glycan analysis in the past decade. However, current isotopic tags for glycan labeling are mostly mass-shift tags generating mass differences in precursor ions for quantification, which can complicate mass spectra. In this study, we report the synthesis and characterization of isobaric aldehyde reactive tags (iARTs) for glycan quantification using tandem MS. We applied iARTs to the relative identification and quantification of glycans of gp120, a glycoprotein from human immunodeficiency virus. The results show that iARTs provide strong signals for glycan identification. Although we only show the synthesis and characterization of two iARTs reagents, iARTs can be readily expanded to six-plex tags for quantitative analysis of six samples concurrently.
Avocado/soy unsaponifiable (ASU) components are reported to have a chondroprotective effect by virtue of anti-inflammatory and proanabolic effects on articular chondrocytes. The identity of the active component(s) remains unknown. In general, sterols, the major component of unsaponifiable plant material have been demonstrated to be anti-inflammatory in vitro and in animal models. These studies were designed to clarify whether the sterol content of ASU preparations were the primary contributors to biological activity in articular chondrocytes. ASU samples were analyzed by high pressure liquid chromatography (HPLC) and GC mass spectrometry. The sterol content was normalized between diverse samples prior to in vitro testing on bovine chondrocytes. Anabolic activity was monitored by uptake of 35-sulfate into proteoglycans and quantitation of labeled hydroxyproline and proline content after incubation with labeled proline. Anti-inflammatory activity was assayed by measuring reduction of interleukin-1 (IL-1)-induced synthesis of PGE2 and metalloproteases and release of label from tissue prelabeled with S-35.All ASU samples exerted a similar time-dependent up-regulation of 35-sulfate uptake in bovine cells reaching a maximum of greater than 100% after 72 h at sterol doses of 1–10 μg/ml. Non-collagenous protein (NCP) and collagen synthesis were similarly up-regulated. All ASU were equally effective in dose dependently inhibiting IL-1-induced MMP-3 activity (23–37%), labeled sulfate release (15–23%) and PGE2 synthesis (45–58%). Up-regulation of glycosaminoglycan and collagen synthesis and reduction of IL-1 effects in cartilage are consistent with chondroprotective activity. The similarity of activity of ASU from diverse sources when tested at equal sterol levels suggests sterols are important for biologic effects in articular chondrocytes.
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.
Proteomics analysis is important for characterizing tissues to gain biological and pathological insights, which could lead to the identification of disease-associated proteins for disease diagnostics or targeted therapy. However, tissues are commonly embedded in optimal cutting temperature compound (OCT) or they are formalin-fixed and paraffin-embedded (FFPE) in order to maintain tissue morphology for histology evaluation. Although several tissue proteomics analyses have been performed on FFPE tissues using advanced mass spectrometry technologies, high throughput proteomic analysis of OCT-embedded tissues has been difficult due to the interference of OCT in the mass spectrometry analysis. In addition, molecules other than proteins present in tissues further complicate tissue proteomic analysis. Herein, we report the development of a method using Chemical Immobilization of Proteins for Peptide Extraction (CIPPE). In this method, proteins are chemically immobilized onto a solid support; interferences from tissues and OCT embedding are removed by extensive washing of proteins conjugated on the solid support. Peptides are then released from the solid phase by proteolysis, enabling mass spectrometry analysis. This method was first validated by eliminating OCT interference from a standard protein, human serum albumin, where all the unique peaks contributed by OCT contamination were eradicated. Finally, this method was applied for the proteomic analysis of frozen and OCT-embedded tissues using iTRAQ labeling and 2D liquid chromatography tandem mass spectrometry. The data showed reproducible extraction and quantitation of 10,284 proteins from 3,996 protein groups and a minimal impact of OCT embedding on the analysis of the global proteome of the stored tissue samples.
New technologies in mass spectrometry are beginning to mature and show unique advantages for the identification and quantitation of proteins. In recent years, one of the significant goals of clinical proteomics has been to identify biomarkers that can be used for clinical diagnosis. As technology has progressed, the list of potential biomarkers has grown. However, the verification and validation of these potential biomarkers is increasingly challenging and require high-throughput quantitative assays, targeting specific candidates. Targeted proteomics bridges the gap between biomarker discovery and the development of clinically applicable biomarker assays.
We collected serial blood samples from children in the intensive care unit who underwent daily bathing with 2% chlorhexidine gluconate (CHG)-impregnated cloths. Low concentrations of CHG were detected in a few blood samples, indicating absorption through intact skin. There was no suggestion that CHG accumulated in the blood with repeated exposures.
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