Mark W. Duncan

Quantitative matrix-assisted laser desorption/ionization mass spectrometry

This review summarizes the essential characteristics of matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOF MS), especially as they relate to its applications in quantitative analysis. Approaches to quantification by MALDI-TOF MS are presented and published applications are critically reviewed.

The pros and cons of peptide-centric proteomics

Recommendations on how best to exploit the strengths of peptide-centric proteomics and avoid its pitfalls.

Quantitative and qualitative differences in protein expression between papillary thyroid carcinoma and normal thyroid tissue

In order to better understand basic mechanisms of tumor development and identify potential new biomarkers, we have performed difference gel electrophoresis (DIGE) and peptide mass fingerprinting on pooled protein extracts from patients with papillary thyroid carcinoma (PTC) compared with matched normal thyroid tissue. Image analysis of DIGE gels comparing PTC and matched normal thyroid tissue protein indicated that 25% of the protein spots were differentially expressed at a 2.5‐fold cutoff and 35% at two‐fold. Comparison between two different pools of protein from normal thyroid tissues revealed differential protein expression of only 4% at 2.5‐fold and 6% at two‐fold cutoff. One hundred ninety‐two protein spots were identified by MALDI‐TOFMS, representing 90 distinct proteins. Excluding albumin, globins and thyroglobulin, imaging software determined 31 proteins to be differentially expressed at the two‐fold (or greater) level. Individual gel comparisons (PTC vs. matched normal) from five patients established that 15/31 (48%) of these proteins exhibited statistically significant differential expression. Previously identified molecular markers in this group of proteins include cathepsin B, cytokeratin 19, and galectin‐3. Novel differentially expressed proteins include S100A6, moesin, HSP70 (BiP), peroxiredoxin 2, protein phosphatase 2, selenium binding protein 1, vitamin D binding protein, and proteins involved in mitochondrial function. The use of two‐dimensional gel electrophoresis (2DGE) revealed a significantly altered protein mass and/or pI in 10%–15% of proteins, suggesting alternatively spliced forms and other posttranslational modification of proteins revealed by this approach. We confirmed S100A6 as a potentially useful biomarker using immunohistochemical analysis (85% sensitivity and 69% specificity for distinguishing benign from malignant thyroid neoplasms). In summary, proteomic analysis of PTC using DIGE and mass spectrometry has confirmed several known biomarkers, uncovered novel potential biomarkers, and provided insights into global pathophysiologic changes in PTC. Many of the differences observed would not have been detected by genomic or other proteomic approaches.

Proteomics as a tool for clinically relevant biomarker discovery and validation

The excitement associated with clinical applications of proteomics was initially focused on its potential to serve as a vehicle for both biomarker discovery and drug discovery and routine clinical sample analysis. Some approaches were thought to be able to “identify” mass spectral characteristics that distinguished between control and disease samples, and thereafter it was believed that the same tool could be employed to screen samples in a high-throughput clinical setting. However, this has been difficult to achieve, and the early promise is yet to be fully realized. While we see an important place for mass spectrometry in drug and biomarker discovery, we believe that alternative strategies will prove more fruitful for routine analysis. Here we discuss the power and versatility of 2D gels and mass spectrometry in the discovery phase of biomarker work but argue that it is better to rely on immunochemical methods for high-throughput validation and routine assay applications.

Discovery and validation of protein abundance differences between follicular thyroid neoplasms.

Distinguishing between benign follicular thyroid adenoma (FTA) and malignant follicular thyroid carcinoma (FTC) by cytologic features alone is not possible. Molecular markers may aid distinguishing FTA from FTC in patients with indeterminate cytology. The aim of this study is to define protein abundance differences between FTC from FTA through a discovery (proteomics) and validation (immunohistochemistry) approach. Difference gel electrophoresis (DIGE) and peptide mass fingerprinting were performed on protein extracts from five patients with FTC and compared with six patients with FTA. Individual gel comparisons (i.e., each FTC extract versus FTA pool) were also performed for the five FTC patients. Immunohistochemical validation studies were performed on three of the identified proteins. Based on DIGE images, 680 protein spots were matched on individual gels. Of these, 102 spots showed statistically significant differences in abundance between FTC and FTA in the individual gel analyses and were therefore studied further. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used to identify 54 of these protein spots. Three candidates involved in protein folding (heat shock protein gp96, protein disulfide isomerase A3, and calreticulin) were studied by immunohistochemistry. Moderate calreticulin immunohistochemical staining was the best single marker with a high negative predictive value (88%); combining all three markers (any marker less than moderate staining) had the best positive predictive value (75%) while still retaining a good negative predictive value (68%). With DIGE, we identified 54 proteins differentially abundant between FTC and FTA. Three of these were validated by immunohistochemistry. These findings provide further insights into the diagnosis, prognosis, and pathophysiology of follicular-derived thyroid neoplasms.

Plasma Protein Profiling of Mild Cognitive Impairment and Alzheimer's Disease Across Two Independent Cohorts

To unlock the full potential of disease modifying treatments, it is essential to develop early biomarkers for Alzheimers disease (AD). For practical reasons, blood-based markers that could provide a signal at the stage of mild cognitive impairment (MCI) or even earlier would be ideal. Using the proteomic approach of isobaric tagging for relative and absolute quantitation (iTRAQ), we compared the plasma protein profiles of MCI, AD, and cognitively normal control subjects from two independent cohorts: the Sydney Memory and Ageing Study (261 MCI subjects, 24 AD subjects, 411 controls) and the Hunter Community Study (180 MCI subjects, 153 controls). The objective was to identify any proteins that are differentially abundant in MCI and AD plasma in both cohorts, since they might be of interest as potential biomarkers, or could help direct future mechanistic studies. Proteins representative of biological processes relevant to AD pathology, such as the complement system, the coagulation cascade, lipid metabolism, and metal and vitamin D and E transport, were found to differ in abundance in MCI. In particular, levels of complement regulators C1 inhibitor and factor H, fibronectin, ceruloplasmin, and vitamin D-binding protein were significantly decreased in MCI participants from both cohorts. Several apolipoproteins, including apolipoprotein AIV, B-100, and H were also significantly decreased in MCI. Most of these proteins have previously been reported as potential biomarkers for AD; however, we show for the first time that a significant decrease in plasma levels of two potential biomarkers (fibronectin and C1 inhibitor) is evident at the MCI stage.

Assessment of post‐mortem‐induced changes to the mouse brain proteome

This study was designed to assess the influence of high‐energy head‐focused microwave irradiation and the post‐mortem interval on measurements of the mouse brain proteome. Difference gel electrophoresis was used to compare mouse brain protein levels in animals killed by decapitation, where the tissue was held at 25°C for selected time intervals post‐mortem, and by high‐energy head‐focused microwave irradiation followed by immediate resection. Microwave‐mediated killing was used because it comprehensively snap‐inactivates enzymes while largely retaining brain cytoarchitecture. Of the 912 protein spots common to at least eight of 10 gels analyzed, 35 (3.8%) showed significant differences in levels (t‐test; p < 0.05) depending on whether animals were killed by microwave irradiation or decapitation. When animals were killed by decapitation, 43 protein spots (4.7%) showed changes in levels over the post‐mortem interval (anova; p < 0.05). The vast majority of the near 1000 proteins evident on a 2D gel were stable for up to 4 h. These data have important implications for studies of proteins in the brain, whether based on analysis of tissue derived from animal models or from humans.

Biomarkers in rheumatology, now and in the future

This review examines the biomarker development process by using rheumatic disorders as the disease model for discussion. We evaluate the current role of biomarkers in the practice of rheumatology and discuss their likely role in the future. We define the essential components of the biomarker development pipeline and discuss the issue of fitness for purpose, i.e. what the biomarker(s) might offer in a clinical setting. As a component of this review we also highlight several emerging technologies that are beginning to provide practical solutions to support biomarker validation. In the process, we highlight some scenarios where additional biomarkers would add considerable value to clinical practice, and we review appropriate methods for each. We also emphasize some important but infrequently discussed considerations, including the need for protein variant verification. Ultimately, the adroit application of the methods of proteomics will transform the practice rheumatology and allow personalized clinical practice to become a reality.

Circulating Cytokines and Growth Factors in Pediatric Pulmonary Hypertension

Background. Management of pediatric pulmonary hypertension (PH) remains challenging. We have assessed a panel of circulating proteins in children with PH to investigate their value as predictive and/or prognostic biomarkers. From these determinations, we aim to develop a practical, noninvasive tool to aid in the management of pediatric PH. Methods. Twelve cytokines and growth factors putatively associated with lung or vascular disease were examined in plasma specimens from 70 children with PH using multiplex protein array technology. Associations between hemodynamics, adverse events, and protein markers were evaluated. Results. Epidermal growth factor (EGF) and IL-6 were associated with important hemodynamics. Of the twelve proteins, VEGF and IL-6 were significantly, univariately associated with the occurrence of an adverse event, with odds ratios (95% confidence intervals) of 0.56 (0.33–0.98) and 1.69 (1.03–2.77), respectively. When hemodynamic predictors were combined with protein markers, the ability to predict adverse outcomes within the following year significantly increased. Conclusions. Specific circulating proteins are associated with hemodynamic variables in pediatric PH. If confirmed in additional cohorts, measurement of these proteins could aid patient care and design of clinical trials by identifying patients at risk for adverse events. These findings also further support a role for inflammation in pediatric PH.

Proteomic analysis of mature adipo cytes from obese patients in relation to aging

Obesity and aging are interrelated conditions that both cause changes in adipocyte metabolism and affect the distribution of fat in both subcutaneous and visceral depots. In addition, both weight gain and aging can lead to similar clinical outcomes such as insulin resistance, cardiovascular disease, type 2 diabetes mellitus, atherosclerosis and stroke. Our objective was to examine the changes in protein expression within the subcutaneous adipose tissue of obese patients, matched for BMI, in relation to age. Mature adipocytes were isolated from liposuction samples of abdominal subcutaneous adipose tissue collected from both young (26.2±4.3 (mean age±SD); n=7) and old (52.2±4.7 (mean age±SD); n=7) obese individuals. Total protein extracts were then compared by two-dimensional difference in gel electrophoresis (2D DIGE). Thirty differentially expressed protein spots (ANOVA test, p≤0.05; fold-change ≥1.8) were detected, of which, 15 were identified by MALDI-TOF mass spectrometry. These were comprised of a total of thirteen unique protein sequences. Nine proteins were more abundant in the adipocytes isolated from old vs. young individuals. These proteins included prohibitin 1, protein disulphide isomerase A3, beta actin, profilin, aldo-ketoreductase 1 C2, alpha crystallin B and the annexins A1, A5 and A6. Four other proteins were less abundant in the adipocytes from old, obese subjects and these included keratin type 2 cytoskeletal 1, keratin type 2 cytoskeletal 10 and hemoglobins A and B. The differentially abundant proteins were investigated by Ingenuity Pathway Analysis (IPA) to reveal their associations with known biological functions. This analysis identified signal transducer and activator of transcription 3 as the central molecule in the connectivity map and the apoptotic pathway as the pathway with the highest score. Differences in the abundances of several proteins were confirmed by immunoblotting: i.e., prohibitin 1, protein disulphide isomerase A3, beta actin, profilin and signal transducer and activator of transcription 3 proteins. In conclusion, proteomic analysis of subcutaneous adipose tissue reveals differences in the abundance of proteins in adipocytes isolated from young vs. old individuals. These differentially abundant proteins are involved in the regulation of apoptosis, cellular senescence and inflammatory response. All these are common pathologic events in both obesity and aging.