Proteomics: technology development and applications

Abstract

Technology development in, and the application of, proteomics are emerging areas among chemical engineers and others who presented at the 2008 American Institute of Chemical Engineers (AIChE) Annual Meeting. Overall, the centennial meeting offered a broad current perspective on the discipline of chemical engineering as it enters its second century. Biomedical and biochemical engineering continue to grow as important facets of the discipline. Within these, the value and applicability of proteomics were demonstrated in a number of interesting presentations. This year, as in the recent past, the AIChE Annual Meeting was held in conjunction with the American Electrophoresis Society Annual Meeting. American Electrophoresis Society presenters offered further academic and industrial viewpoints on the still-developing role of proteomics and proteomic technologies in biological and clinical analyses.

The focus of chemical engineers on biological problems continues to grow and evolve. This was exceedingly evident at the 2008 American Institute of Chemical Engineers (AIChE) Annual Meeting held in Philadelphia, PA, USA. No fewer than 150 unique sessions over the 5-day meeting focused on some aspect of biomedical or biochemical engineering. Moreover, the honorary Institute Lecture was given by Mark E Davis (California Institute of Technology, CA, USA), whose work, similar to that of three of the past four Institute Lecturers, focuses on biological problems. In particular, he emphasized that the commitment of chemical engineers should be on the burgeoning field of ‘molecular engineering’, the understanding and manipulation of molecules and the molecular-scale properties of materials. It is at this molecular scale that many of the biological sessions were focused. These ranged across many topics, including biofuels, biosensors, nanobiotechnology, high-throughput analyses, gene and drug delivery, genomics and proteomics. The sessions also included those from the annual meeting of the American Electrophoresis Society (AES), which has been meeting jointly with the AIChE since 2001.

A number of sessions had a specific focus on proteomics, such as ‘Genomics and Proteomics for Tissues and Organs’, ‘Proteomics and Metabolic Approaches to Systems Biology’, ‘Advances in Proteomics: New Technologies’ (AES session) and ‘Advances in Proteomic Analysis and Microfluidic Technologies’ (AES session). Presentations from these sessions, as well as other relevant presentations, will be highlighted in this report. In cases where the breadth of the meeting precluded our attending presentations, we have summarized accepted abstracts that we feel would be of interest to the reader. The technical programs and presentation abstracts for the meetings are available online [101].

Our discussion is focused on two thematic areas – application of proteomic methodologies and the development of new technologies to improve existing proteomic techniques. In the former category, proteomic applications to a wide variety of biological problems were discussed. Zeyu Sun (Advisor: Kenneth Reardon, Colorado State University, CO, USA) presented on the impact of phytoestrogen exposure on the proteome of cardiomyocytes as measured by 2DE. The motivation for the work was the markedly reduced incidence of cardiovascular disease among populations with high soy diets. Using cultured cells treated with moderate (1 µM) and high (50 µM) doses of the soy-derived phytoestrogen genistein, they used 2DE and mass spectrometry (MS) to analyze proteins isolated through hydrophobic and hydrophilic extraction. While the group continues to explore the functional roles of the dozens of differentially expressed proteins, they discussed one protein, voltage-dependent anion-selective channel (VDAC) 2, which showed a threefold upregulation in response to moderate doses of genistein and a twofold downregulation to high doses of genistein. They hypothesize that this protein, which is only expressed in cardiac tissue, may improve cardiac health through the prevention of apoptosis and enhancement of the cellular energy state. A mechanism that would explain the different response to moderate and high levels of genistein is still being explored. Brett Chromy (Lawrence Livermore National Laboratory, CA, USA) spoke on the development and use of proteomic technologies for investigating host–pathogen interactions [1]. This presentation focused on using 2D difference gel electrophoresis, SELDI-MS and protein arrays for profiling the proteome of the pathogen Yersinia pestis in different infection models.

The function of proteins in the proteome depends on their physical and chemical properties. Ke Xia (Advisor: Wilfredo Colon, Rensselaer Polytechnic Institute, NY, USA) described a technique, diagonal 2DE, for identifying kinetically stable proteins (KSPs), proteins that are especially recalcitrant to denaturation [2]. The technique begins with normal 1D sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, with the exception that the samples are exposed to SDS for a limited time and not boiled prior to loading. Proteins of weak structure are denatured by SDS and run normally on the gel. KSPs run more slowly due to the reduced complexation with SDS. After the first dimension, a lane on the gel is excised and boiled in buffer and then placed horizontally along the top of a second gel for a second size separation. After boiling, all proteins, including the KSPs, are denatured. Thus, the second gel has a stripe of proteins along the diagonal that were denatured in both steps and a number of proteins below the diagonal that ran more quickly in the second step than the first. These proteins below the diagonal are the putative KSPs. In the Escherichia coli proteome, enzymes (70% of KSPs vs 30% of total proteins) and highly multimeric (more than four subunits) complexes (33 vs 8%, respectively) were especially over-represented among 50 unique putative KSPs. This is understandable given the importance of enzymes in cell function, and hence the need for their greater stability. Highly multimeric complexes would also be expected to possess enhanced stability through direct interactions among the various subunits.

Examining another aspect of protein structure, Christine Carag (Advisor: Dennis Discher, University of Pennsylvania, PA, USA) discussed the use of the ‘Cys shotgun’ labeling technique developed in her research group [3,4]. Briefly, the technique provides a means of assessing cytoskeletal changes induced by force through the parallel labeling of all available cysteine residues in the intracellular proteome by thiol-reactive labels, such as 5-({2-[(iodoacetyl)amino]ethyl}amino)naphthalene-1-sulfonic acid (IAEDANS). The system on which this talk focused was the cytoskeletal organization of red blood cells, with a particular focus on spectrin proteins. In their native structure, spectrins have buried cysteine residues that can be exposed upon the application of force to the cell. The presentation described some of the recent work they have done in using the approach to determine the forces required to alter spectrin structure and to assess the co-operativity of this structural alteration.

Proteomic technology development presentations were primarily focused on sample preparation and fractionation. Ning Bao (Advisor: Chang Lu, Purdue University, IN, USA) spoke on the development of a microfluidic device for extraction and concentration of intracellular proteins from bacteria without using chemical or biological reagents [5]. A microscale bead array was used to capture small populations of bacteria, yielding up to 10⁴-fold concentration in 40 min. The trapped cells were then lysed using electrical pulses, and the proteins released were detected downstream. The functioning of this device was demonstrated using green fluorescent protein as the model intracellular protein, and the potential for reuse of the device was also discussed.

Tom Berkelman (Bio-Rad Laboratories, Inc., CA, USA) presented on the enriching and separation of low-abundance proteins in complex samples using ProteoMiner™ technology. The presentation described a nontargeted approach for depleting higher abundance proteins while enriching lower abundance proteins in samples where the proteins span a large dynamic range. The ProteoMiner technique uses a bead-based combinatorial peptide library of ligands to bind proteins in a nontargeted manner. The technique is predicated on the principle that, for a limited number of binding sites, high-abundance proteins rapidly saturate the binding capacity of their ligands while low-abundance proteins do not. This results in a majority of the high-abundance proteins being unable to bind and washing out, whereas a larger fraction of the low-abundance proteins remain bound until eluted off the beads. Thus, this approach simultaneously depletes high-abundance proteins and enriches low-abundance proteins from complex protein samples. The effectiveness of ProteoMiner in enriching low-abundance plasma proteins was demonstrated using leaf protein extracts, which are dominated by the large and small subunits of Rubisco. The technique was shown to improve the resolution of the proteome in samples extracted by both native and denaturing techniques; in the denaturing case, 23 new proteins were identified that were not detectable in control samples.

Electrophoretic and microfluidic techniques for sample preparation were also discussed. Bingwen Lu (Advisor: Cornelius Ivory, Washington State University, WA, USA) described multiplexing different orthogonal separation schemes (analogous to 2DE) into a single microfluidic platform. Specifically, a polydimethyl siloxane device was used to preconcentrate low-abundance proteins initially using isotachophoresis, followed by isoelectric focusing to separate proteins further based on their charge. The authors also discussed integrating a third dimension into the platform for more effective purification of low-abundance proteins. Yu-Wen Huang (Advisor: Victor Ugaz, Texas A&M University, TX, USA) spoke on the development of a generic platform for the detection of charged analytes, including proteins. In this technology, the detection of a compacted film of microbubbles produced due to the electrolysis of water was used for detecting electrophoretically captured charged analytes. A key feature of this approach is that the microbubbles are visible under white light and enable detection of proteins in solution without the use of labels or dyes.

As can be seen from the breadth of the presentations described here, the contributions of chemical engineers to the discipline of proteomics occur at the level of technology development, quantitative analysis, sample preparation and new applications. The evolution of proteomics in practice will continue to benefit from the unique perspective that molecular engineers can offer. Moreover, it is also evident that the collaborative efforts of engineers with clinicians and life scientists will further speed the development and translation of important technologies and findings to the most critical applications. It is expected that the further scale and cost improvements required for widespread clinical and laboratory use of proteomics will probably arise from such collaborative efforts.

Financial & competing interests disclosure

The authors gratefully acknowledge support for this work from the US NIH (RR024439 to SP Walton) and the American Heart Association (0755112Y to A Jayaraman). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.