Meeting Report

10.1586/14789450.5.3.385 © 2008 Expert Reviews Ltd ISSN 1478-9450 385www.expert-reviews.com

24th Annual Meeting of the American Electrophoresis SocietyExpert Rev. Proteomics 5(3), 385–387 (2008)

David E GarfinAmerican Electrophoresis Society, 1563 Solano Avenue, #341, Berkeley, CA 94707, USATel: +1 510 526 0874Fax: +1 510 526 2206degarfin@sonic.net

24th Annual Meeting of the American Electrophoresis SocietyNovember 5–9, 2007, Salt Lake City, UT, USA

Presentations at the 2007 meeting of the American Electrophoresis Society covered manyaspects of this key separation technology. In total there were three plenary speakers, twoinvited talks, 85 technical talks and 14 posters in a 5-day meeting. The three plenary speakerspresented their work with each of them discussing somewhat different multiplexedproteomics approaches. The invited speakers discussed ways to improve resolution andshorten running times in proteomic and genomic separations. The proteomics technical talksdescribed applications of 1D and 2D gel electrophoresis, capillary electrophoresis and micro-scale platforms. This report is limited to a small number of those presentations that discussedproteomics directly.

Proteomics was well represented at the 24thAnnual Meeting of the American Electrophore-sis Society [101]. The meeting was held in SaltLake City, Utah from November 5–9 2007 asTopical Conference T3 within the larger 99thMeeting of the American Institute of ChemicalEngineers [102]. The meeting was organized byJoseph Biernacki (Tennessee Technical Univer-sity, TN, USA) and Wayne Patton (Enzo LifeSciences, NY, USA). This review provides onlya small sampling of the talks bearing directly onproteomics. A review of presentations on micro-devices is planned for a future edition of theonline journal Biomicrofluidics.

Three plenary speakers spoke about variousaspects of proteomics. Carol Giometti(Argonne National Laboratory, IL, USA)described multiplexed studies of the bacteriumShewanella oneidensis. The organism, whichbinds and reduces various metals, especiallyuranium, is being considered for use in bio-remediation [1,2]. The Giometti group uses both2DE and 2D-LC-MS/MS to inventory pro-teins expressed by the bacterium grown in high-and low-iron media. Over 500 proteins havebeen identified using 2DE-MS/MS, with anadditional 500 identified by 2D-LC-MS/MS.Membrane proteins are localized on outer or

inner membrane surfaces by biotinylation ofouter membrane proteins and collected by avi-din affinity chromatography. Phosphopeptideanalyses are performed by western blotting.Metalloproteins are determined by x-ray fluores-cence induced in a synchrotron. Katheryn Res-ing (University of Colorado, CO, USA) is a pro-ponent of shotgun proteomics [3]. Shedemonstrated a 2DE study of differentiationinduced in megakaryocytes by phorbol esters.2DE yielded approximately 150 spots of inter-est, many at very low abundance. Protein identi-fication was possible after pooling togethermatching spots extracted from 20 different gels.Most of Resing’s work is with MudPIT. Hergroup uses an approach to data analysis thatintegrates results of searches with Sequest andMascot and employs new prediction algorithms.They presented a description of a shotgun pro-tocol used on melanoma cells that combinesscanning for phosphate loss with parallel proteinprofiling. The results suggest that most of thephosphorylation sites (∼5000) in the melanomaproteome are being sampled because thenumber is consistent with estimates that approx-imately a third of proteins are phosphorylatedand that the average phosphoprotein has aboutthree phosphorylation sites. David Friedman

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(Vanderbilt University, TN, USA) uses DIGE technology fordifferential analyses [4]. He commented that many large-scaleproteomics experiments are underpowered and give too manyfalse positives because not enough repetitions are performed.Friedman addresses this problem with multiple samples usingthree replicate gels each. Of concern are the three main sourcesof variation in a sample:

• Technical noise caused during sample preparation andfractionation

• Biological noise arising from normal variation in biologicalsamples (particularly problematic in clinical samples)

• Variance of the biological system itself

It is generally agreed that repetition is the only way toaccount for these variations. Statistical analyses such asunsupervised hierarchical clustering and principle componentanalysis can be applied to complex datasets and can helpaddress the sources of variation.

Two invited speakers reported work aimed at improving theresolution and speed of electrophoretic separations; work that issure to have bearing on proteomics research in the future.Victor Ugaz (Texas A&M University, TX, USA) describedstudies aimed at optimizing rapid, high-resolution separationsof DNA oligonucleotides in microdevices. A specially designedchamber contains crosslinked polyacrylamide-filled channelsand incorporates arrays of electrodes, heaters and temperaturesensors. Individually addressable electrode arrays can be used tosequentially concentrate, focus, meter and inject DNA into anelectrophoresis channel. Single- and double-stranded oligo-nucleotides approximately 140–360 monomers long were stud-ied. Cornelius Ivory (Washington State University, WA, USA)presented an invited talk outlining the approaches his group istaking to better understand and control the various causes ofdispersion in separation media in general, and in microchannelsin particular. Specialized devices are being used to investigatedispersive mechanisms.

Sessions covered wide-ranging topics from fundamental elec-trokinetics to advanced apparatus designs to sophisticated pro-teomic and genomic analyses. Cornelius Ivory described severalequilibrium gradient focusing methods [5]. All use opposing,orthogonal physical or chemical forces to establish steady-stateresolution of appropriate molecules. At present, isoelectricfocusing (IEF) is the only such method in wide use. Gradientsof electric field, conductivity, temperature and mobility can (atleast theoretically) be made to produce separations similar toIEF. Yue Ge (US Environmental Protection Agency, NC, USA)described 2DE studies of arsenic-induced oxidative stress inmouse kidneys and human keratinocytes. Standard methodswere employed showing that specific toxic effects of arsenicexposure can be fingerprinted with 2DE. Presentations on envi-ronmental proteomics included work presented by CarlaLacerda (Colorado State University, Colorado, USA) on theresponse of soil microbes to cadmium stress. Both 2DE andshotgun methods showed significant shifts in protein regulation

in the presence of cadmium in a strain of the highly cadmium-resistant bacterium Burkholderia cepacia. A unique aspect ofthis work was the use of stable isotope labeling (14N/15N or12C/13C) for differential analysis. Clear differences were foundin the proteomes of cultures grown in low and high cadmiumlevels, including proteins involved in energy metabolism,defense and transport. Interestingly, there was little overlapbetween proteins identified by gel and shotgun analyses. Thegroup has more confidence in the identifications made with theshotgun method than with gels. A similar environmental pro-teomics study was presented by Ana Pereira-Medrano (Univer-sity of Sheffield, UK) who described a differential analysis alsousing isotopic labeling (14N/15N) of the proteome of Pedobactercryoconitis, a microorganism from Alpine glaciers that canmetabolize and degrade petroleum hydrocarbons. The organ-ism was grown at two different temperatures and with two dif-ferent carbon sources. Proteins were separated by 2DE andgiven the usual treatments for peptide mass fingerprinting. Pep-tides were validated by searching the spectra for 15N doubletpeaks at the proper mass-to-charge ratio positions based on thenumber of nitrogen atoms in the unlabeled peptide.

Tom Berkelman (Bio-Rad Laboratories, Inc., CA, USA) gavean instructive presentation describing his development of thein-gel fluorescent protein stain named Flamingo™. The dyehas binding characteristics similar to those of SDS and destain-ing is unnecessary since unbound dye does not fluoresce. Thedye fluoresces in the visible wavelength range, with excitationand emission/detection wavelengths of 508 and 535–560 nm,respectively. The detection limit of Flamingo is given as 30 pgof protein and linear quantification over 4 orders of magnitudeis claimed. The stain gives good coverage in peptide massfingerprinting by MALDI, presumably because Flamingo doesnot appear to oxidize tryptophan residues as do some otherstains. Aaron Sin (Protein Forest, Inc., MA, USA) described thecompany’s IEF prefractionation device. This is a variant ofother commercial IEF devices in that a stack of gel plugs each ata specific pH is placed with the long axis perpendicular to theelectric field. Proteins from complex mixtures separate into theplug with a pH nearest to the protein’s isoelectric point. Up tofour samples can be run concurrently. Separation is said to beaccomplished in 30 min. An orthogonal affinity capture anddetection strategy for identifying and characterizing phospho-proteins was presented by Alvydas Mikulskis (PerkinElmer Life& Analytical Sciences, MA, USA). The method does not useantibodies, but utilizes coordination chemistries similar to thoseused in immobilized metal ion affinity chromatography(IMAC) approaches. Dinuclear zinc complexes are used both toisolate phosphoproteins and to locate them in gels. Phospho-protein bands are cut from gels and enzymatically digested.Phosphopeptides are purified by affinity capture on titaniumdioxide-coated magnetic beads, eluted and characterized bymass spectrometry (MS). Nancy Kendrick (Kendrick Laborato-ries, Inc., WI, USA) spoke about a method for reciprocal affin-ity depletion for isolating disease-specific proteins. Antibodies

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were raised from proteins obtained from normal and diseasedrat lungs, and columns were made from purified antibodies.Proteins from normal animals were passed over columns ofantibodies against the proteins from the diseased animals andvice versa. This gave two populations of proteins; one specificfor normal tissue and the other for diseased tissue. Phospho-proteins were located on immunoblots of 2DE separationswith antiphosphotyrosine antibody (PY20). Soumya Keshava-murthy (Mississippi State University, MS, USA) describedprogress in ongoing studies of the behavior of red blood cellsin dielectrophoresis chambers. Since the movement of bloodcells in dielectrophoretic fields is characteristic of their ABOtype, it might be possible to devise a diagnostic microdevicebased on these findings. Benjamin Hawkins (Cornell Univer-sity, NY, USA) also spoke about dielectrophoresis. Alterationsof channel geometries are being investigated in the attempt todevelop a tool for screening for different mycobacterium spe-cies. Ryan Forster (Northwestern University, IL, USA)described tests on a device for removing proteins, but notnucleic acids, from biological fluids. Block copolymers ofacrylamide and dihexylacrylamide bind molecules containinghydrophobic patches such as proteins. DNA passes throughthe system and is suitable for sequencing.

Three student posters received awards. First, second andthird prizes were won by Kaela Leonard (Mississippi State Uni-versity), Jennifer Anne Pascal (Tennessee Technical University)and Yusuke Tatsumi (Kyoto Institute of Technology, Kyoto,Japan) for posters on dielectrophoresis cells, electrophoresis in aCouette-type chamber, and the dynamics of silver staining in2DE gels, respectively.

The 2008 AES Annual Meeting will be held in Philadelphia,USA, November 17–21 [101,102]. It is being organized by ShashiMurthy (Northeastern University, MA, USA) and JonathanPosner (Arizona State University, AZ, USA) and will cover thedesign of and applications of separation devices from nano- tomacro-scale, as well as including substantial reportage of proteomicmethods and applications. The meeting is open to all.

Financial & competing interests disclosure

The author has no relevant affiliations or financial involvement with anyorganization or entity with a financial interest in or financial conflictwith the subject matter or materials discussed in the manuscript. Thisincludes employment, consultancies, honoraria, stock ownership or options,expert testimony, grants or patents received or pending, or royalties.

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

References

1 Giometti CS, Khare T, Tollaksen SL et al. Analysis of the Shewanella oneidensis proteome by two-dimensional gel electrophoresis under nondenaturing conditions. Proteomics 3 (5), 777–785 (2003).

2 Kolker E, Picone AF, Galperin MY et al. Global profiling of Shewanella oneidensis MR-1: expression of hypothetical genes and improved functional annotations. Proc. Natl Acad. Sci. USA 102 (6), 2099–2104 (2005).

3 Ruth MC, Old WM, Emrick MA et al. Analysis of membrane proteins from human chronic myelogenous leukemia cells: comparison of extraction methods for multidimensional LC-MS/MS. J. Proteome Res. 5(3), 709–719 (2006).

4 Friedman DB, Wang SE, Whitwell CW, Caprioli RM, Arteaga CL. Multivariable difference gel electrophoresis and mass spectrometry. Mol. Cell. Proteomics 6(1), 150–169 (2007).

5 Ivory CF. Alternative electrofocusing methods. In: Handbook of Isoelectric Focusing and Proteomics. Garfin D, Ahuja S (Eds). Elsevier, Amsterdam, The Netherlands 297–319 (2005).

Websites

101 American Electrophoresis Societywww.aesociety.org

102 American Institute of Chemical Engineerswww.aiche.org

Affiliation

• David E Garfin, PhDPresident of the American Electrophoresis Society, 1563 Solano Avenue, #341, Berkeley, CA 94707, USATel: +1 510 526 0874Fax: +1 510 526 2206degarfin@sonic.net