As evidenced by Thons data, the correlation betweenproteomic techniques is not particularly good. This obser-vation is not unexpected because each technique has itsstrengths and weaknesses in detecting certain types ofproteins and in quantifying protein expression. Mem-brane protein changes were not reliably detected usinggel-based technology, but this is a known limitation of atechnique which has other drawbacks including poordynamic range and insensitivity to both hydrophobic pro-teins and extremes of isoelectric point. In general, thetechniques correlate well when one starts with simplegroups of proteins, but are quickly overwhelmed whenmore complex protein mixtures are studied like thosefound in whole-cell lysates. One would hope that PLTs aremore amenable to proteomic studies, being simpler thancells that contain nuclei and other complex organelles.
The study by Thon and coworkers further demon-strates that independent information can be gleaned byusing distinct proteomic techniques, and because of tech-nical characteristics, it may not be fair to directly comparethem. Although it may not yet be time to abandon gels forPLT proteome research, the data of Thon and coworkerssuggest that 2D gels, even when combined with sensitivefluorescent DIGE dyes, may not be sufficient to determinechanges in the PLT proteome with storage. Again, the PLTproteome is very complex and one is often interested inproteins that are of less abundance. For example, mito-chondria appear to play a role in the PLT storage defect(PSD), but are found in such low numbers in PLTs thatsmall yet significant changes in PLT mitochondrial pro-teins might remain obscure amid more abundant PLTproteins.
Which of the proteomic technologies should thetransfusion community focus on to better understand thePSD? Other researchers struggle with this question, butThons group appear to have had success with the iTRAQapproach where an impressive number of proteins wereidentified. Indeed, other investigators have pointed outthe potential superiority of this peptide-centric approachfor studying PLT proteins.6 Depending on the sensitivityand throughput of the mass spectrometer used, peptide-centric approaches might be better than gels for detectingless abundant proteins. They should also bemore capableof identifying hydrophobic membrane proteins. Otherevents on the horizon that may facilitate PLT proteomicefforts include the development of: 1) remarkably sensi-tivemass spectrometers that can identify proteins startingwith minuscule amounts of peptide, 2) enhanced liquidchromatographic and other separation techniques thatare being directly interfaced with mass spectrometers, 3)other clever quantitative and solution-based proteomicstechniques, and 4) a dizzying array of sophisticatedhybrid mass spectrometers that combine analyzers ofdifferent types. Finally, one must not overlook the criticalrole of bioinformatic tools to the success of any proteom-
ics study.7 In many ways, advances in proteomic bioinfor-matics have not kept pace with the technical advances inmass spectrometersfurther progress will benefit all.
Numerous tests have been proposed to monitor forPLT storagerelated effects. Themost useful ones are thosethat correlate with PLT survival upon transfusion. Doesthe study by Thon and colleagues provide clues as to themost relevant proteins associated with the PSD? For pro-teomic technology to be useful in studying this age-oldproblem, protein variables will need to correlate with theone true test of PLT quality, surviving posttransfusion in ahemostatically active state. It is unlikely that the merepresence or absence of a PLT protein will correlate withPLT efficacy. The proteins identified as potential markersof the PSD included b-actin, septin 2, and gelsolin. Theseare relatively abundant PLT proteins that proteomic tech-niques should detect and quantify in a reproduciblemanner. Other abundant proteins that appear to changewith storage such as fibrinogen, 14-3-3, and a glucose-dependent transporter may actually be serving as surro-gate markers for PLT activation. More likely, PLT viabilitywill depend on quantitative differences in a combinationof PLT proteins, a group that may include lower abun-dance proteins. If in fact a single or small number of PSDbiomarkers are identified that correlate with PLT efficacy,then other more established techniques for quantifyingproteins could be developed that are more amenable towidespread PLTmonitoring. By leveraging the strengths ofproteomics in detecting alterations in the PLT proteomewith storage, novel quality assurance tools could thenevolve to monitor PLT products.
Regardless of the proteomic method employed, alter-ations in PLT proteins occurring during storage may beattributed to a variety ofmechanisms. Changes associatedwith the PSD should occur when proteins are modifiedbecause proteins are largely responsible for the structureand function of all cells including PLTs. The changes maybe a direct effect of collection and/or storage ormay be anindirect effect. An example of the latter is the decreasein PLT pH during storage. PLT proteins also 1) degradeduring storage,8 2) translocate from the PLT interior to thePLT membrane (e.g., CD62P), or 3) shift from PLT storagegranules to the cytosolic region.
Similar to other tests used to check PLT quality (e.g.,swirling), proteomic studies are also difficult to replicate;despite similar protocols, proteomic labs will report dif-fering results based on the equipment and informaticstechniques used. Depending on the confidence level andlevel of significance selected for protein identification,labs will identify more or less proteins. Thon and cowork-ers selected a p value of less than 0.01 as significant. Thisvaluewhich is too low for a genomics experiment wherethousands of genes are testedmay be appropriate fora proteomics experiment. Unfortunately, standards forprotein identification cutoffs have not been defined.
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Depending on how peptide data (in this case, mass-to-charge ratios) is analyzed and which protein database isutilized for protein identification, interlaboratory differ-ences are expected. These and other realities will hamperstandardization of proteomic-based studies and theirdevelopment as a quality assurance tool.
Advances are also being made in other gel-free pro-teomic techniques that might add to the armamentariumfor studying the PSD in addition to iTRAQ and ICAT.Although higher numbers of proteins are generally iden-tified by ICAT and iTRAQ than by gels, even the mostadvanced proteomic techniques available may be subop-timal for detecting changes in the PLT proteome duringstorage. With further progress in proteomics, other areasof transfusion medical practice could be addressed,including pathogen inactivation, blood substitutes, andhematopoietic growth factors.9 For example, proteomicstechniques could be used to screen compounds that inac-tivate viruses and bacteria while monitoring for PLTprotein alterations. For now, Thon and colleagues arrive atthe logical conclusion that a combination of protein- andpeptide-centric approaches should be used to study thePSD.
Peter L. Perrotta, MDDepartment of PathologyWest Virginia UniversityHealth Sciences Center
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