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1.  Quantifying proteins by mass spectrometry: The selectivity of SRM is only part of the problem 
Proteomics  2009;9(5):1124-1127.
Precise and accurate protein quantification is critical to many areas of proteomics. Antibody-based approaches are costly and time-consuming to develop, consequently, there is considerable interest in alternative quantitative methods that are versatile and can be implemented without the considerable delays associated with antibody development and characterization. Approaches based on MS have therefore attracted considerable attention and are now frequently touted as the most practical and powerful of all options. Nevertheless, there are serious limitations associated with quantifying a protein based on tandem mass analysis of one or two peptides generated by either chemical or enzymatic cleavage. In an accompanying Viewpoint article, Molloy and coworkers point out that selectivity is not necessarily guaranteed despite the power of SRM. Here we address an additional concern that can also compromise specificity. In complex mammalian systems, multiple proteins can serve as precursors of a single peptide and consequently, depending on the peptide(s) selected, protein levels may be significantly under- or overestimated.
PMCID: PMC4166569  PMID: 19253279
Mass spectrometry; Protein quantification; Selected reaction monitoring
2.  Alpha-Linolenic Acid: An Omega-3 Fatty Acid with Neuroprotective Properties—Ready for Use in the Stroke Clinic? 
BioMed Research International  2015;2015:519830.
Alpha-linolenic acid (ALA) is plant-based essential omega-3 polyunsaturated fatty acids that must be obtained through the diet. This could explain in part why the severe deficiency in omega-3 intake pointed by numerous epidemiologic studies may increase the brain's vulnerability representing an important risk factor in the development and/or deterioration of certain cardio- and neuropathologies. The roles of ALA in neurological disorders remain unclear, especially in stroke that is a leading cause of death. We and others have identified ALA as a potential nutraceutical to protect the brain from stroke, characterized by its pleiotropic effects in neuroprotection, vasodilation of brain arteries, and neuroplasticity. This review highlights how chronic administration of ALA protects against rodent models of hypoxic-ischemic injury and exerts an anti-depressant-like activity, effects that likely involve multiple mechanisms in brain, and may be applied in stroke prevention. One major effect may be through an increase in mature brain-derived neurotrophic factor (BDNF), a widely expressed protein in brain that plays critical roles in neuronal maintenance, and learning and memory. Understanding the precise roles of ALA in neurological disorders will provide the underpinnings for the development of new therapies for patients and families who could be devastated by these disorders.
PMCID: PMC4350958  PMID: 25789320
3.  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.
PMCID: PMC2722264  PMID: 19106161
quantification; quantitative analysis; MALDI; mass spectrometry; biomarkers
5.  Proteomics-Based Disease Biomarkers 
PMCID: PMC3205713  PMID: 22110951
6.  Quantitative and Qualitative Differences in Protein Expression Between Papillary Thyroid Carcinoma and Normal Thyroid Tissue† 
Molecular carcinogenesis  2006;45(8):613-626.
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
PMCID: PMC1899163  PMID: 16788983
thyroid cancer; proteomics; molecular markers; DIGE
7.  The 2S albumin allergens of Arachis hypogaea, Ara h 2 and Ara h 6, are the major elicitors of anaphylaxis and can effectively desensitize peanut-allergic mice 
Clinical and Experimental Allergy  2012;42(2):326-336.
Ara h 2 and Ara h 6, co-purified together in a 13-25 kD fraction (Ara h 2/6; 20 kD fraction) on gel filtration chromatography, account for the majority of effector activity in a crude peanut extract (CPE) when assayed with RBL SX-38 cells sensitized with IgE from human peanut allergic sera.
To determine if Ara h 2/6 are the primary peanut allergens responsible for allergic reactions in vivo and to determine if Ara h 2/6 would be sufficient to prevent allergic reactions to a complete CPE.
An oral sensitization mouse model of peanut allergy was used to assess the activity of Ara h 2/6 (20 kD) and CPE without the 20 kD fraction (CPE w/o 20 kD) for allergic provocation challenge and immunotherapy. The activity of these preparations was also tested in an assay of histamine release from human basophils in whole blood.
Compared to mice challenged with control CPE, mice challenged with CPE w/o 20 kD experienced reduced symptoms (p<0.05) and a smaller decrease in body temperature (p<0.01). Results with the basophil histamine release assay corroborated these findings (p<0.01). The mouse model was also used to administer Ara h 2/6 (20 kD) in an immunotherapy protocol, in which peanut-allergic mice treated with the 20 kD fraction experienced significantly reduced symptoms, changes in body temperature, and mast cell protease (MMCP-1) release compared to placebo (p<0.01 for all parameters). Importantly, immunotherapy with the 20 kD fraction was just as effective as treatment with CPE, whereas CPE w/o 20 kD was significantly less effective for higher dose peanut challenges.
Conclusions and Clinical Relevance
Ara h 2/6 are the most potent peanut allergens in vivo and can be used to desensitize peanut-allergic mice. These results have potential implications for clinical research in the areas of diagnosis and immunotherapy for peanut allergy.
PMCID: PMC3270336  PMID: 22288514
Food allergy; peanut allergy; Ara h 2; Ara h 6; desensitization; immunotherapy; human basophil assay; mouse model
8.  Vitamin D binding protein isoforms as candidate predictors of disease extension in childhood arthritis 
Journal of Proteomics  2012;75(17):5479-5492.
Juvenile idiopathic arthritis (JIA) comprises a poorly understood group of chronic autoimmune diseases with variable clinical outcomes. We investigated whether the synovial fluid (SF) proteome could distinguish a subset of patients in whom disease extends to affect a large number of joints.
SF samples from 57 patients were obtained around time of initial diagnosis of JIA, labeled with Cy dyes and separated by two-dimensional electrophoresis. Multivariate analyses were used to isolate a panel of proteins which distinguish patient subgroups. Proteins were identified using MALDI-TOF mass spectrometry with expression verified by immunochemical methods. Protein glycosylation status was confirmed by hydrophilic interaction liquid chromatography.
A truncated isoform of vitamin D binding protein (VDBP) is present at significantly reduced levels in the SF of oligoarticular patients at risk of disease extension, relative to other subgroups (p < 0.05). Furthermore, sialylated forms of immunopurified synovial VDBP were significantly reduced in extended oligoarticular patients (p < 0.005).
Reduced conversion of VDBP to a macrophage activation factor may be used to stratify patients to determine risk of disease extension in JIA patients.
Graphical abstract
PMCID: PMC3443749  PMID: 22771520
Juvenile idiopathic arthritis; Proteomics; Synovial fluid; Vitamin D binding protein; Inflammation
9.  Purification and Structural Characterization of Siderophore (Corynebactin) from Corynebacterium diphtheriae 
PLoS ONE  2012;7(4):e34591.
During infection, Corynebacterium diphtheriae must compete with host iron-sequestering mechanisms for iron. C. diphtheriae can acquire iron by a siderophore-dependent iron-uptake pathway, by uptake and degradation of heme, or both. Previous studies showed that production of siderophore (corynebactin) by C. diphtheriae is repressed under high-iron growth conditions by the iron-activated diphtheria toxin repressor (DtxR) and that partially purified corynebactin fails to react in chemical assays for catecholate or hydroxamate compounds. In this study, we purified corynebactin from supernatants of low-iron cultures of the siderophore-overproducing, DtxR-negative mutant strain C. diphtheriae C7(β) ΔdtxR by sequential anion-exchange chromatography on AG1-X2 and Source 15Q resins, followed by reverse-phase high-performance liquid chromatography (RP-HPLC) on Zorbax C8 resin. The Chrome Azurol S (CAS) chemical assay for siderophores was used to detect and measure corynebactin during purification, and the biological activity of purified corynebactin was shown by its ability to promote growth and iron uptake in siderophore-deficient mutant strains of C. diphtheriae under iron-limiting conditions. Mass spectrometry and NMR analysis demonstrated that corynebactin has a novel structure, consisting of a central lysine residue linked through its α- and ε- amino groups by amide bonds to the terminal carboxyl groups of two different citrate residues. Corynebactin from C. diphtheriae is structurally related to staphyloferrin A from Staphylococcus aureus and rhizoferrin from Rhizopus microsporus in which d-ornithine or 1,4-diaminobutane, respectively, replaces the central lysine residue that is present in corynebactin.
PMCID: PMC3326035  PMID: 22514641

Results 1-9 (9)