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1.  Quantitative Plant Phosphoproteomics 
Current Opinion in Plant Biology  2011;14(5):507-511.
Protein phosphorylation is a major post-translational modification in plants crucial for the regulation of diverse cellular functions. In the early stages of this field, efforts focused on the qualitative detection, identification, and cataloging of in vivo protein phosphorylation sites. Recently these studies have advanced into utilizing quantitative mass spectrometric measurements, capable of dynamically monitoring changes in phosphorylation levels in response to genetic and environmental alterations. This review will highlight current untargeted and targeted mass spectral technologies used for quantitative phosphoproteome measurements in plants, and provide a discussion of these phosphorylation changes in relation to important biological events.
doi:10.1016/j.pbi.2011.06.008
PMCID: PMC3253357  PMID: 21764629
2.  Quantitative improvements in peptide recovery at elevated chromatographic temperatures from μLC/MS analyses of brain using SRM mass spectrometry 
Analytical chemistry  2010;82(9):3435-3440.
Elevated chromatographic temperatures are well recognized to provide beneficial analytical effects. Previously, we demonstrated that elevated chromatographic temperature enhances the identification of hydrophobic peptides prepared from enriched membrane samples. Here, we quantitatively assess and compare the recovery of peptide analytes from both simple and complex tryptic peptide matrices using the SRM mass spectrometry. Our study demonstrates that elevated chromatographic temperature results in significant improvements in the magnitude of peptide recovery for both hydrophilic and hydrophobic peptides from both simple and complex peptide matrices. Importantly, the analytical benefits for quantitative measurements in whole mouse brain matrix are demonstrated, suggesting broad utility in the proteomic analyses of complex mammalian tissues. Any improvement in peptide recovery from chromatographic separations translates directly to the apparent sensitivity of downstream mass analysis in μLC-MS/MS based proteomic applications. Therefore, the incorporation of elevated chromatographic temperatures should result in significant improvements in peptide quantification as well as detection and identification.
doi:10.1021/ac100359p
PMCID: PMC2903057  PMID: 20373813
3.  Quantitative strategies to fuel the merger of discovery and hypothesis-driven shotgun proteomics 
The ultimate goal of most shotgun proteomic pipelines is the discovery of novel biomarkers to direct the development of quantitative diagnostics for the detection and treatment of disease. Differential comparisons of biological samples identify candidate peptides that can serve as proxys of candidate proteins. While these discovery approaches are robust and fairly comprehensive, they have relatively low throughput. When merged with targeted mass spectrometry, this pipeline can fuel hypothesis-driven studies and the development of novel diagnostics and therapeutics.
doi:10.1093/bfgp/elp008
PMCID: PMC3105289  PMID: 19398505
quantitative shotgun proteomics; biomarker discovery; targeted mass spectrometry; human tissue
4.  MudPIT Analysis: Application to Human Heart Tissue 
Although two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) has been used as the standard proteomic approach for separating proteins in a complex mixture, this technique has many drawbacks. These include a limited molecular mass range, poor separation of highly acidic or basic proteins, and exclusion of the majority of membrane proteins from analysis. Considering the important functions of many membrane proteins, such as receptors, ion transporters, signal transducers, and cell adhesion proteins, it is increasingly important that these proteins are not excluded during the global proteomic analysis of cellular systems. Multidimensional Protein Identification Technology (MudPIT) offers a gel-free alternative to 2D-PAGE for the analysis of both membrane and soluble proteins.
The goal of this chapter is to provide detailed methods for using MudPIT to profile both membrane and soluble proteins in complex unfractionated samples. Methods discussed will include tissue homogenization, sample preparation, MudPIT, data analysis, and an application for the analysis of unfractionated total tissue homogenate from human heart.
doi:10.1007/978-1-60327-310-7_20
PMCID: PMC2765120  PMID: 19153700
MudPIT; 2D-PAGE; proteomics; membrane proteins; human heart explants
5.  High Quality Catalog of Proteotypic Peptides from Human Heart 
Journal of proteome research  2008;7(11):5055-5061.
Proteomics research is beginning to expand beyond the more traditional shotgun analysis of protein mixtures to include targeted analyses of specific proteins using mass spectrometry. Integral to the development of a robust assay based on targeted mass spectrometry is prior knowledge of which peptides provide an accurate and sensitive proxy of the originating gene product (i.e., proteotypic peptides). To develop a catalog of “proteotypic peptides” in human heart, TRIzol extracts of left-ventricular tissue from nonfailing and failing human heart explants were optimized for shotgun proteomic analysis using Multidimensional Protein Identification Technology (MudPIT). Ten replicate MudPIT analyses were performed on each tissue sample and resulted in the identification of 30 605 unique peptides with a q-value ≤ 0.01, corresponding to 7138 unique human heart proteins. Experimental observation frequencies were assessed and used to select over 4476 proteotypic peptides for 2558 heart proteins. This human cardiac data set can serve as a public reference to guide the selection of proteotypic peptides for future targeted mass spectrometry experiments monitoring potential protein biomarkers of human heart diseases.
doi:10.1021/pr800239e
PMCID: PMC2765113  PMID: 18803417
proteotypic peptides; targeted mass spectrometry; human heart explant; dilated cardiomyopathy; MudPIT

Results 1-5 (5)