Alexander Lazarev

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.

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.

Separation methods in proteomics

PART I SAMPLE PREPARATION Applications of Pressure Cycling Technology (PCT) in 2DGE F. Tao, J. Behnke, C. Li, C. Saravis, R.T. Schumacher, and N.P. Lawrence Applications of Ion-Exchange Chromatography (IEX) to Reduce Sample Complexity Prior to Two-Dimensional Gel Electrophoresis (2DGE) M.G. Pluskal, E. Golenko, and M.F. Lopez Use of Camelid Antibody Fragments in the Depletion and Enrichment of Human Plasma Proteins for Proteomics Applications B. Dawson, P. Hermans, and M. ten Haaft High-Throughput Plasma Depletion with Chicken Antibodies for Proteomic Analysis S.W. Tam, L. Huang, D. Hinerfeld, D. Innamorati, X. Fang, W.W. Zhang, J. Pirro, and J.S. Feitelson Immunoaffi nity Depletion of High-Abundant Proteins for Proteomic Sample Preparation N. Zolotarjova, B.Boyes, J. Martosella, L.S. Yang, G. Nicol, K. Zhang, C. Szafranski, and J. Bailey Isolation of Plasma Membrane Proteins for Proteomic Analysis C. Fenselau and A. Rahbar New Ultrafiltration and Solid Phase Extraction Techniques Improve Serum Peptide Detection E. Chernokalskaya, S. Gutierrez, A.M. Pitt, A.V. Lazarev, and J.T. Leonard PART II SAMPLE PREFRACTIONATION AND ANALYSES Tools for Sample Preparation and Prefractionation in Two-Dimensional Gel Electrophoresis A. Posch, A. Paulus, and M.G. Brubacher Optic Nerve Fractionation for Proteomics S.K. Bhattacharya, J. S. Crabb, S.P. Annangudi, K.A. West, X. Gu, J. Sun, V.L. Bonilha, G. Smejkal, K. Shadrach, J.G. Hollyfield, and J.W. Crabb Fractionation of Retina for Proteomic Analyses S.K. Bhattacharya, K.A. West, X. Gu, J.S. Crabb, K.Renganathan, Z. Wu, J. Sun, and J.W. Crabb Reducing Protein Sample Complexity with Free Flow Electrophoresis (FFE) A. Kuchumov, G. Weber, and C. Eckerskorn PART III APPLICATIONS OF ELECTROPHORESIS IN PROTEOMICS Destreaking Strategies for Two-Dimensional Electrophoresis F. Bai, S. Liu, and F.A. Witzmann Proteomic Approaches to the Study of Rheumatoid Arthritis M. Antonovici, K. Dasuri, H. El-Gabalawy, and J.A. Wilkins Immunoglobulin Patterns in Health and Disease I. Miller and M. Goldfarb Difference Gel Electrophoresis (DIGE) M. Unlu and J. Minden Principles and Challenges of Basic Protein Separation by Two-Dimensional Electrophoresis A. Posch, A. Paulus, and M.G. Brubacher Multidimensional Separation of Membrane Proteins S. Francis-McIntyre and S.J. Gaskel Structural Approaches to Glycoproteomics H. Geiser, C. Silvescu, and V. Reinhold Enrichment and Analysis of Glycoproteins in the Proteome N.L. Wilson, N.G. Karlsson, and N.H. Packer PART IV APPLICATIONS OF HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY Proteomic Analyses Using High-Efficiency Separations and Accurate Mass Measurements J.M. Jacobs and R.D. Smith Middle-Out Proteomics: Incorporating Multidimensional Protein Fractionation and Intact Protein Mass Analysis as Elements of a Proteomic Analysis Workflow S.J. Berger, A.B. Chakraborty, K. Millea, H. Liu, and S.A. Cohen Polymeric Monolithic Capillary Columns in Proteomics A.R. Ivanov PART V RELATED TECHNIQUES Staining Proteins in Polyacrylamide Gels G. Smejkal Multiplexed Proteomics: Fluorescent Detection of Proteins, Glycoproteins, and Phospoproteins in Two-Dimensional (2D) Gels B. Schulenberg Direct Immunoprobing on Glyoxyl Agarose Composite Gels J.R. Shainoff Nuclear Magnetic Resonance-Driven Chemical Proteomics: The Functional and Mechanistic Complement to Proteomics P.K. Pullela and D.S. Sem Electrophoretic Nuclear Magnetic Resonance: Toward High-Throughput Structural Characterization of Biological Signaling Processes Q. He and X. Song Index

Sample preparation in biological mass spectrometry

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Method Development for Fecal Lipidomics Profiling

Robust methodologies for the analysis of fecal material will facilitate the understanding of gut (patho)physiology and its role in health and disease and will help improve care for individual patients, especially high-risk populations, such as premature infants. Because lipidomics offers a biologically and analytically attractive approach, we developed a simple, sensitive, and quantitatively precise method for profiling intact lipids in fecal material. The method utilizes two separate, complementary extraction chemistries, dichloromethane (DCM) and a methyl tert-butyl ether/hexafluoroisopropanol (MTBE) mixture, alone or with high pressure cycling. Extracts were assessed by liquid chromatography-high-resolution mass spectrometry-based profiling with all ion higher energy collisional dissociation fragmentation in both positive and negative ionization modes. This approach provides both class-specific and lipid-specific fragments, enhancing lipid characterization. Solvents preferentially extracted lipids based on hydrophobicity. More polar species preferred MTBE; more hydrophobic compounds preferred DCM. Pressure cycling differentially increased the yield of some lipids. The platform enabled analysis of >500 intact lipophilic species with over 300 lipids spanning 6 LIPID MAPS categories identified in the fecal matter from premature infants. No previous report exists that provides these data; thus, this study represents a new paradigm for assessing nutritional health, inflammation, and infectious disease in vulnerable populations.

Isolation of functional mitochondria from rat kidney and skeletal muscle without manual homogenization

Isolation of functional and intact mitochondria from solid tissue is crucial for studies that focus on the elucidation of normal mitochondrial physiology and/or mitochondrial dysfunction in conditions such as aging, diabetes, and cancer. There is growing recognition of the importance of mitochondria both as targets for drug development and as off-target mediators of drug side effects. Unfortunately, mitochondrial isolation from tissue is generally carried out using homogenizer-based methods that require extensive operator experience to obtain reproducible high-quality preparations. These methods limit dissemination, impede scale-up, and contribute to difficulties in reproducing experimental results over time and across laboratories. Here we describe semiautomated methods to disrupt tissue using kidney and muscle mitochondria preparations as exemplars. These methods use the Barocycler, the PCT Shredder, or both. The PCT Shredder is a mechanical grinder that quickly breaks up tissue without significant risk of overhomogenization. Mitochondria isolated using the PCT Shredder are shown to be comparable to controls. The Barocycler generates controlled pressure pulses that can be adjusted to lyse cells and release organelles. The mitochondria subjected to pressure cycling-mediated tissue disruption are shown to retain functionality, enabling combinations of the PCT Shredder and the Barocycler to be used to purify mitochondrial preparations.

Label-free mass spectrometry-based relative quantification of proteins separated by one-dimensional gel electrophoresis

Here we present a matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI-TOF/TOF)-based label-free relative protein quantification strategy that involves sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation of proteins followed by in-gel trypsin digestion. The main problem encountered in gel-based protein quantification is the difficulty in achieving complete and consistent proteolytic digestion. To solve this problem, we developed a high-pressure-assisted in-gel trypsin digestion method that is based on pressure cycling technology (PCT). The PCT approach performed at least as well as the conventional overnight in-gel trypsin digestion approach in parameters such as number of peaks detected, number of peptides identified, and sequence coverage, and the digestion time was reduced to 45 min. The gel/mass spectrometry (MS)-based label-free protein quantification method presented in this work proved the applicability of the signal response factor concept for relative protein quantification previously demonstrated by other groups using the liquid chromatography (LC)/MS platform. By normalizing the average signal intensities of the three most intense peptides of each protein with the average intensities of spiked synthetic catalase tryptic peptides, which we used as an internal standard, we observed spot-to-spot and lane-to-lane coefficients of variation of less than 10 and 20%, respectively. We also demonstrated that the method can be used for determining the relative quantities of proteins comigrating during electrophoretic separation.

Pressure cycling technology in systems biology.

Systems biologists frequently seek to integrate complex data sets of diverse analytes into a comprehensive picture of an organisms biological state under defined environmental conditions. Although one would prefer to collect these data from the same sample, technical limitations with traditional sample preparation methods often commit the investigator to extracting one type of analyte at the expense of losing all others. Often, volume further constrains the range of experiments that can be collected from a single sample. The practical solution employed to date has been to rely on information collected from multiple replicate experiments and similar historical or reported data. While this approach has been popular, the integration of information collected from disparate single-analyte sample preparation streams increases uncertainty due to nonalignment during comparative analysis, and such gaps accumulate quickly when combining multiple data sets. Regrettably, discontinuities between separate data streams can confound a whole understanding of the biological system being investigated. This difficulty is further compounded for researchers handling highly pathogenic samples, in which it is often necessary to use harsh chemicals or high-energy sterilization procedures that damage the target analytes. Ultra-high pressure cycling technology (PCT), also known as barocycling, is an emerging sample preparation strategy that has distinct advantages for systems biology studies because it neither commits the researcher to pursuing a specific analyte nor leads to the degradation of target material. In fact, samples prepared under pressure cycling conditions have been shown to yield a more complete set of analytes due to uniform disruption of the sample matrix coupled with an advantageous high pressure solvent environment. Fortunately, PCT safely sterilizes and extracts complex or pathogenic viral, bacterial, and spore samples without adversely affecting the constituent biomolecules valued as informative and meaningful analytes. This chapter provides procedures and findings associated with incorporating PCT into systems biology as a new and enabling approach to preanalytical sample treatment.

Thermal stabilization of tissues and the preservation of protein phosphorylation states for two-dimensional gel electrophoresis.

2‐DE is typically capable of discriminating proteins differing by a single phosphorylation or dephosphorylation event. However, a reliable representation of protein phosphorylation states as they occur in vivo requires that both phosphatases and kinases are rapidly and completely inactivated. Thermal stabilization of mouse cerebral cortex homogenates effectively inactivated these enzymes, as evidenced by comparison with unstabilized tissues where abscissal pI shifts were a common feature in 2‐D gels. Of the 588 matched proteins separated on 2‐D gels comparing stabilized and unstabilized tissues, 53 proteins exhibited greater than twofold differences in spot volume (ANOVA, p<0.05). Phosphoprotein‐specific staining was corroborated by the identification of 16 phosphoproteins by nano‐LC MS/MS and phosphotyrosine kinase activity assay.

Pilot Proteomic Profile of Differentially Regulated Proteins in Right Atrial Appendage Before and After Cardiac Surgery Using Cardioplegia and Cardiopulmonary Bypass

Background— Although highly protective, cardiac surgery using cardioplegia and cardiopulmonary bypass (CP/CPB) subjects myocardium to hypothermic reversible ischemic injury that can impair cardiac function which results in a greatly enhanced risk of mortality. Acute changes in myocardial contractile activity are likely regulated via protein modifications. We performed the following study to determine changes in the protein profile of human myocardium following CP/CPB. Methods and Results— Right atrial appendage was collected from 8 male patients pre and post-CP/CPB. Atrial tissue lysates were subjected to 2-dimensional electrophoresis, total protein staining, gel averaging, and quantitative densitometry. Ten prominent spots regulated in response to CP/CPB were identified using mass spectrometry. Two hundred twenty-five and 256 protein spots were reliably detected in 2D-gels from pre- and post-CP/CPB patients, respectively. Five unique (ie, not detected post-CP/CPB) and 17 significantly increased spots were detected pre-CP/CPB. Thirty-four unique and 25 significantly increased spots were detected in the post-CP/CPB group. Identified proteins that changed after CP/CPB included: MLC-2a, ATP-synthase delta chain and Enoyl-CoenzymeA hydratase, glutathione-s-transferase omega, α-1-acid-glycoprotein, and phosphatidylethanolamine-binding protein. Conclusions— Cardiac surgery results in multiple consistent changes in the human myocardial protein profile. CP/CPB modifies specific cytoskeletal, metabolic, and inflammatory proteins potentially involved in deleterious effects of CP/CPB.