Gary B. Smejkal

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

Comparison of in-gel protein separation techniques commonly used for fractionation in mass spectrometry-based proteomic profiling

Fractionation of complex samples at the cellular, subcellular, protein, or peptide level is an indispensable strategy to improve the sensitivity in mass spectrometry‐based proteomic profiling. This study revisits, evaluates, and compares the most common gel‐based protein separation techniques i.e. 1D SDS‐PAGE, 1D preparative SDS‐PAGE, IEF‐IPG, and 2D‐PAGE in their performance as fractionation approaches in nano LC‐ESI‐MS/MS analysis of a mixture of protein standards and mitochondrial extracts isolated from rat liver. This work demonstrates that all the above techniques provide complementary protein identification results, but 1D SDS‐PAGE and IEF‐IPG had the highest number of identifications. The IEF‐IPG technique resulted in the highest average number of detected peptides per protein. The 2D‐PAGE was evaluated as a protein fractionation approach. This work shows that the recovery of proteins and resulting proteolytic digests is highly dependent on the total volume of the gel matrix. The performed comparison of the fractionation techniques demonstrates the potential of a combination of orthogonal 1D SDS‐PAGE and IEF‐IPG for the improved sensitivity of profiling without significant decrease in throughput.

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.

The Application of a Novel Nanovolume Capillary Electrophoresis- Based Protein Analysis System in Personalized & Translational MedicineResearch

There is increasing evidence that abnormal protein synthesis and modification are associated with a variety of human diseases. In the coming era of personalized/precision medicine, it will be required to utilize a rapid, highly sensitive and quantitative method to analyze the proteins and related post-translational modifications in clinical specimens in order to better define specific therapies for patients. However, the current gold standard in proteomic analysis is still the traditional Western blot, which requires many manual steps with lower sensitivity and provides a semi-quantitative read-out. Here, in this manuscript, we present the first report of a novel fully automated Capillary Electrophoresis (CE)-based immunodetection technology, called the Simple Western size assay, which is run on the instrument, called the Simon TM . This technology is based on nanovolume size-based protein separation that can be used to quantify proteomic profiles of clinical specimens for both biomarker discovery and diagnostics. Our results demonstrated that the Simple Western has higher sensitivity of target protein detection, a greater linear dynamic range of different molecular weight proteins, high reproducibility and the capacity for the higherthroughput screening of samples using small sample input volumes compared to traditional Western blot analysis. In addition, the quantitativeness and accuracy, the exquisite sensitivity and reduced background noise, has made the Simon Western highly versatile. This technology can quantitative the level of protein and related post-translational modifications in translational medicine research, such as specific biomarkers for diabetes and cancer research. These results based on several broad applications in this study suggest the Simple Western size assay will be a novel potential protein detection accelerator in the personalized and translational medicine era..

Genomics and proteomics: of hares, tortoises and the complexity of tortoises

The draft human genome sequence was published in 2001 and took 13 years to complete. Necessity being the mother of invention, the accelerated development of DNA sequencing technologies led to the completion of the Human Genome Project nearly a year ahead of schedule [1]. In 1998, the total sequencing output from the entire Human Genome Project was about 200 million base pairs for the year. By January 2003, the Department of Energy Joint Genome Institute had reached the capacity to sequence 1.5 billion bases per month. Today, the Bejing Genome Institute in China has the capacity to generate the equivalent of 10,000 human genomes per year. In 2010, the Bejing Genome Institute generated ten times the amount of data the National Center for Biotechnology Information had generated in the past 20 years. In early 2012, Oxford Nanopore Technologies announced that its secondgeneration GridIons would be able to sequence a human genome in 15 min [2]. Enter proteomics, the proverbial tortoise in the race with the hare.

The meaning of ‘native’

The use of detergents The polyoxyethylene sorbitan series detergents such as Tween 20 and 80 (and the lesser known Tweens 40, 60, 65 and 85) are widely used to prevent protein aggregation and other nonspecific interactions while generally preserving biological activity. However, their high micellar molecular weight complicates their removal from protein solutions. Tween 20 is commonly used at concentrations over six-times its critical micelle concentration where over 85% of the detergent mass exists as 38 kDa micelles [1]. For Triton X–100, the mean aggregate is 143 monomers and the mean micellar molecular weight is over 90 kDa [2]. Further, the binding of detergents can radically change protein molecular mass. For example, the 460 kDa insulin receptor in the presence of Triton X–100 becomes a complex of over 1000 kDa [3]. Cytochrome P-450 exhibits a molecular mass of 100 kDa in CHAPS, but over 300 kDa in sodium cholate [4]. It is both interesting and important to note that lowering the detergent concentration can also effect protein mass. Adenylate cyclase is twice its molecular size in 0.01% Lubrol than it is in 0.1% Lubrol [5]. Is this native? Protease inhibitors & the ‘preservation’ of native state Though considered a preservative, EDTA can actually drive the denaturation or partial denaturation of some proteins. EDTA has been shown to cause irreversible denaturation and aggregation of zinc-binding proteins [6]. Even more disconcerting is the finding that the unfolding of zinc-binding domains by EDTA can lead to non-specific protein-protein interactions [7]. For platelets incubated with EDTA, the irreversible loss of platelet aggregation and a diminished capacity to bind fibrinogen, corresponding to modification of the membrane glycoproteins has been reported [8]. PMSF and AEBSF covalently bind to serines in the active sites of serine proteases. Because it is so stable, AEBSF can continue to modify proteins for several days, where it may non-specifically bind and modify Tyr, Lys, His and N-terminal residues [9]. Are these native proteins?

Revisiting Jurassic Park: The Isolation of Proteins from Amber Encapsulated Organisms Millions of Years Old

Dominican Republic amber from the Oligo-Miocene epoch, 20–40 million years ago, was interrogated for residual protein. Tandem mass spectrometric analysis of trypsin digests of proteins from two silver-stained bands excised from SDS-PAGE led to the identification of 84 peptides from 19 Saccharomyces proteins. All peptides were identified from one high molecular weight gel band, suggesting a high degree of cross-linking of these proteins. This study reports the first experimental data on the identification of prehistoric proteins from amber.

Two-Dimensional Gel Electrophoresis Reveals Differential Protein Expression Between Individual Daphnia

Analysis of individual genetic variation is paramount to understanding how organisms and communities respond to changes in the environment and requires a model system with well-developed molecular resources and a solid foundation of ecological knowledge. Traditional genetic model systems (E. coli, yeast, fly, worm, and mouse) have served as workhorses in elucidating virtually all of the knowledge in modern molecular biology. While these systems were chosen for their robustness in laboratory studies, virtually nothing is known about their life histories in their native environment. By contrast, newmodel systems, which have typically been studied in depth, from an ecological perspective are severely limited in regards to their molecular resources.