Mary F. Lopez

Background-free, high sensitivity staining of proteins in one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gels using a luminescent ruthenium complex

SYPRO Ruby dye is a permanent stain comprised of ruthenium as part of an organic complex that interacts noncovalently with proteins. SYPRO Ruby Protein Gel Stain provides a sensitive, gentle, fluorescence‐based method for detecting proteins in one‐dimensional and two‐dimensional sodium dodecyl sulfate‐polyacrylamide gels. Proteins are fixed, stained from 3h to overnight and then rinsed in deionized water or dilute methanol/acetic acid solution for 30 min. The stain can be visualized using a wide range of excitation sources commonly used in image analysis systems including a 302 nm UV‐B transilluminator, 473 nm second harmonic generation (SHG) laser, 488 nm argon‐ion laser, 532 nm yttrium‐aluminum‐garnet (YAG) laser, xenon arc lamp, blue fluorescent light bulb or blue light‐emitting diode (LED). The sensitivity of SYPRO Ruby Protein Gel Stain is superior to colloidal Coomassie Brilliant Blue (CBB) stain or monobromobimane labeling and comparable with the highest sensitivity silver or zinc‐imidazole staining procedures available. The linear dynamic range of SYPRO Ruby Protein Gel stain extends over three orders of magnitude, which is vastly superior to silver, zinc‐imidazole, monobromobimane and CBB stain. The fluorescent stain does not contain superfluous chemicals (formaldehyde, glutaraldehyde, Tween‐20) that frequently interfere with peptide identification in mass spectrometry. While peptide mass profiles are severely altered in protein samples prelabeled with monobromobimane, successful identification of proteins by peptide mass profiling using matrix‐assisted laser desorption/ionization mass spectrometry was easily performed after protein detection with SYPRO Ruby Protein Gel stain.

A comparison of silver stain and SYPRO Ruby Protein Gel Stain with respect to protein detection in two-dimensional gels and identification by peptide mass profiling

Proteomic projects are often focused on the discovery of differentially expressed proteins between control and experimental samples. Most laboratories choose the approach of running two‐dimensional (2‐D) gels, analyzing them and identifying the differentially expressed proteins by in‐gel digestion and mass spectrometry. To date, the available stains for visualizing proteins on 2‐D gels have been less than ideal for these projects because of poor detection sensitivity (Coomassie blue stain) or poor peptide recovery from in‐gel digests and mass spectrometry (silver stain), unless extra destaining and washing steps are included in the protocol. In addition, the limited dynamic range of these stains has made it difficult to rigorously and reliably determine subtle differences in protein quantities. SYPRO Ruby Protein Gel Stain is a novel, ruthenium‐based fluorescent dye for the detection of proteins in sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) gels that has properties making it well suited to high‐throughput proteomics projects. The advantages of SYPRO Ruby Protein Gel Stain relative to silver stain demonstrated in this study include a broad linear dynamic range and enhanced recovery of peptides from in‐gel digests for matrix assisted laser desorption/ionization‐time of flight (MALDI‐TOF) mass spectrometry.

Lin28a transgenic mice manifest size and puberty phenotypes identified in human genetic association studies

Recently, genome-wide association studies have implicated the human LIN28B locus in regulating height and the timing of menarche. LIN28B and its homolog LIN28A are functionally redundant RNA-binding proteins that block biogenesis of let-7 microRNAs. lin-28 and let-7 were discovered in Caenorhabditis elegans as heterochronic regulators of larval and vulval development but have recently been implicated in cancer, stem cell aging and pluripotency. The let-7 targets Myc, Kras, Igf2bp1 and Hmga2 are known regulators of mammalian body size and metabolism. To explore the function of the Lin28–Let-7 pathway in vivo, we engineered transgenic mice to express Lin28a and observed in them increased body size, crown-rump length and delayed onset of puberty. Investigation of metabolic and endocrine mechanisms of overgrowth in these transgenic mice revealed increased glucose metabolism and insulin sensitivity. Here we report a mouse that models the human phenotypes associated with genetic variation in the Lin28–Let-7 pathway.

High-throughput profiling of the mitochondrial proteome using affinity fractionation and automation

Recent studies have demonstrated the need for complementing cellular genomic information with specific information on expressed proteins, or proteomics, since the correlation between the two is poor. Typically, proteomic information is gathered by analyzing samples on two‐dimensional gels with the subsequent identification of specific proteins of interest by using trypsin digestion and mass spectrometry in a process termed peptide mass fingerprinting. These procedures have, as a rule, been labor‐intensive and manual, and therefore of low throughput. The development of automated proteomic technology for processing large numbers of samples simulataneously has made the concept of profiling entire proteomes feasible at last. In this study, we report the initiation of the (eventual) complete profile of the rat mitochondrial proteome by using high‐throughput automated equipment in combination with a novel fractionation technique using minispin affinity columns. Using these technologies, approximately one hundred proteins could be identified in several days. In addition, separate profiles of calcium binding proteins, glycoproteins, and hydrophobic or membrane proteins could be generated. Because mitochondrial dysfunction has been implicated in numerous diseases, such as cancer, Alzheimers disease and diabetes, it is probable that the identification of the majority of mitochondrial proteins will be a beneficial tool for developing drug and diagnostic targets for associated diseases.

A comparison of cultural characteristics and infectivity of Frankia isolates from root nodules of Casuarina species

The isolations of three new strains ofFrankia were made from root nodules ofCasuarina cunninghamiana growing aeroponically. Two strains, HFPCCI1 and HFPCcI2 isolated by Lopez are typicalFrankia strains, producing sporangia among filamentous mats in culture and, in the absence of combined nitrogen, forming vesicles and showing acetylene reduction. They are red-pigmented and, although failing to nodulateCasuarina hosts, effectively nodulatedElaeagnus andHippophae. A third strain HFPCcI3 isolated by Zhang from the same source, also a typicalFrankia, can form sporangia and vesicles in culture and reduce acetylene, is unpigmented, fails to nodulateElaeagnus but effectively nodulatesC. cunninghamiana andC. equisetifolia. Comparisons are made among all of theCasuarina isolates in our collection from around the world (twelve in all) with regard to their cultural characteristics and capacity to infect host plant species. Questions are raised about the specificity of the various isolates and their possible affinities. Opportunities are suggested for inoculation of seedlings for forestry and field application using the infective, effective strains now available.

An improved formulation of SYPRO Ruby protein gel stain: Comparison with the original formulation and with a ruthenium II tris (bathophenanthroline disulfonate) formulation

SYPRO Ruby protein gel stain is compatible with a variety of imaging platforms since it absorbs maximally in the ultraviolet (280 nm) and visible (470 nm) regions of the spectrum. Dye localization is achieved by noncovalent, electrostatic and hydrophobic binding to proteins, with signal being detected at 610 nm. Since proteins are not covalently modified by the dye, compatibility with downstream proteomics techniques such as matrix‐assisted laser desorption/ionisation‐time of flight mass spectrometry is assured. The principal limitation of the original formulation of SYPRO Ruby protein gel stain, is that it was only compatible with a limited number of gel fixation procedures. Too aggressive a fixation protocol led to diminished signal intensity and poor detection sensitivity. This is particularly apparent when post‐staining gels subjected to labeling with other fluorophores such as Schiffs base staining of glycoproteins with fluorescent hydrazides. Consequently, we have developed an improved formulation of SYPRO Ruby protein gel stain that is fully compatible with commonly implemented protein fixation procedures and is suitable for post‐staining gels after detection of glycoproteins using the green fluorescent Pro‐Q Emerald 300 glycoprotein stain or detection of β‐glucuronidase using the green fluorescent ELF 97 β‐D‐glucuronide. The new stain formulation is brighter, making it easier to manually excise spots for peptide mass profiling. An additional benefit of the improved formulation is that it permits staining of proteins in isoelectric focusing gels, without the requirement for caustic acids.

Selected Reaction Monitoring–Mass Spectrometric Immunoassay Responsive to Parathyroid Hormone and Related Variants

BACKGROUND Parathyroid hormone (PTH) assays able to distinguish between full-length PTH (PTH1-84) and N-terminally truncated PTH (PTH7-84) are of increasing significance in the accurate diagnosis of endocrine and osteological diseases. We describe the discovery of new N-terminal and C-terminal PTH variants and the development of selected reaction monitoring (SRM)-based immunoassays specifically designed for the detection of full-length PTH [amino acid (aa)1-84] and 2 N-terminal variants, aa7-84 and aa34-84. METHODS Preparation of mass spectrometric immunoassay pipettor tips and MALDI-TOF mass spectrometric analysis were carried out as previously described. We used novel software to develop SRM assays on a triple-quadrupole mass spectrometer. Heavy isotope-labeled versions of target peptides were used as internal standards. RESULTS Top-down analysis of samples from healthy individuals and renal failure patients revealed numerous PTH variants, including previously unidentified aa28-84, aa48-84, aa34-77, aa37-77, and aa38-77. Quantitative SRM assays were developed for PTH1-84, PTH7-84, and variant aa34-84. Peptides exhibited linear responses (R(2) = 0.90-0.99) relative to recombinant human PTH concentration limits of detection for intact PTH of 8 ng/L and limits of quantification of 16-31 ng/L depending on the peptide. Standard error of analysis for all triplicate measurements was 3%-12% for all peptides, with <5% chromatographic drift between replicates. The CVs of integrated areas under the curve for 54 separate measurements of heavy peptides were 5%-9%. CONCLUSIONS Mass spectrometric immunoassays identified new clinical variants of PTH and provided a quantitative assay for these and previously identified forms of PTH.

Better approaches to finding the needle in a haystack: Optimizing proteome analysis through automation

Recent evidence has demonstrated a lack of correlation between transcriptional profiles and actual protein levels in cells. Proteome analysis has therefore become indispensable and complementary to genomic analysis for an accurate picture of cellular metabolism. Although proteomics is a relatively young discipline, technology for increasing throughput in proteomic projects is rapidly being developed. The operating paradigm in proteome analysis today is a combination of two‐dimensional (2‐D) gel electrophoresis (for protein resolution) with mass spectrometry (for protein identification). All the intermediary steps in the procedure including gel staining, image analysis, protein spot excision, digestion and mass spectrometry can be automated to increase efficiency and save time. This report reviews the current state of the proteomics technology and discusses approaches to enhance the sensitivity of 2‐D gels with fractionation techniques.

Quantitative phosphoproteomic analysis of the STAT3/IL-6/HIF1α signaling network: An initial study in GSC11 glioblastoma stem cells

Initiation and maintenance of several cancers including glioblastoma (GBM) may be driven by a small subset of cells called cancer stem cells (CSCs). CSCs may provide a repository of cells in tumor cell populations that are refractory to chemotherapeutic agents developed for the treatment of tumors. STAT3 is a key transcription factor associated with regulation of multiple stem cell types. Recently, a novel autocrine loop (IL-6/STAT3/HIF1alpha) has been observed in multiple tumor types (pancreatic, prostate, lung, and colon). The objective of this study was to probe perturbations of this loop in a glioblastoma cancer stem cell line (GSC11) derived from a human tumor by use of a JAK2/STAT3 phosphorylation inhibitor (WP1193), IL-6 stimulation, and hypoxia. A quantitative phosphoproteomic approach that employed phosphoprotein enrichment, chemical tagging with isobaric tags, phosphopeptide enrichment, and tandem mass spectrometry in a high-resolution instrument was applied. A total of 3414 proteins were identified in this study. A rapid Western blotting technique (<1 h) was used to confirm alterations in key protein expression and phosphorylation levels observed in the mass spectrometric experiments. About 10% of the phosphoproteins were linked to the IL-6 pathway, and the majority of remaining proteins could be assigned to other interlinked networks. By multiple comparisons between the sample conditions, we observed expected changes and gained novel insights into the contribution of each factor to the IL6/STAT3/HIF1alpha autocrine loop and the CSC response to perturbations by hypoxia, inhibition of STAT3 phosphorylation, and IL-6 stimulation.

Rapid development of sensitive, high-throughput, quantitative and highly selective mass spectrometric targeted immunoassays for clinically important proteins in human plasma and serum

OBJECTIVES The aim of this study was to develop high-throughput, quantitative and highly selective mass spectrometric, targeted immunoassays for clinically important proteins in human plasma or serum. DESIGN AND METHODS The described method coupled mass spectrometric immunoassay (MSIA), a previously developed technique for immunoenrichment on a monolithic microcolumn activated with an anti-protein antibody and fixed in a pipette tip, to selected reaction monitoring (SRM) detection and accurate quantification of targeted peptides, including clinically relevant sequence or truncated variants. RESULTS In this report, we demonstrate the rapid development of MSIA-SRM assays for sixteen different target proteins spanning seven different clinically important areas (including neurological, Alzheimers, cardiovascular, endocrine function, cancer and other diseases) and ranging in concentration from pg/mL to mg/mL. The reported MSIA-SRM assays demonstrated high sensitivity (within published clinical ranges), precision, robustness and high-throughput as well as specific detection of clinically relevant isoforms for many of the target proteins. Most of the assays were tested with bona-fide clinical samples. In addition, positive correlations, (R2 0.67-0.87, depending on the target peptide), were demonstrated for MSIA-SRM assay data with clinical analyzer measurements of parathyroid hormone (PTH) and insulin growth factor 1 (IGF1) in clinical sample cohorts. CONCLUSIONS We have presented a practical and scalable method for rapid development and deployment of MS-based SRM assays for clinically relevant proteins and measured levels of the target analytes in bona fide clinical samples. The method permits the specific quantification of individual protein isoforms and addresses the difficult problem of protein heterogeneity in clinical proteomics applications.