PDFlib PLOP: PDF Linearization, Optimization, Protection

Page inserted by evaluation versionwww.pdflib.com – sales@pdflib.com

E D I T O R I A L

Proteomics: moving from a discovery to a quality assurance tool

Proteomics is an evolving field that encompassesan array of techniques and instruments used toanalyze proteins on a grand scale. At the mostbasic level, proteomic methods are used to iden-

tify proteins (qualitative proteomics) or to determine howmuch of a particular protein is present (quantitative pro-teomics). More sophisticated proteomic techniques areavailable to characterize proteins that are altered throughposttranslational modification or to understand how pro-teins interact with one another within complex biologicnetworks.1

In this issue of TRANSFUSION, Thon and colleagues2

used several distinct proteomics techniques to studychanges in the platelet (PLT) “proteome” that occur whenPLTs are stored under standard conditions for up to7 days. The proteomics methods employed included bothgel-based (two-dimensional gel electrophoresis–differential gel electrophoresis [DIGE]) and solution-phase techniques (isotope-codes affinity tagging [ICAT]and isotope tagging for relative and absolute quantitation[iTRAQ]). The latter are termed “peptide-centric” by theauthors because only small pieces of the protein (<20amino acids long) are analyzed across experimentalsamples. The “protein-centric” gel-based methods sepa-rate proteins by differential mobility in their native state.Although gel-based methods are straightforward, theyremain technically challenging when used to studycomplex protein mixtures.

Thon and colleagues utilized proteomic techniquesthat still have limitations despite being reasonablymature. As might be expected, gel-based techniques werenot particularly sensitive to changes in the PLT proteomewith storage, identifying only a small number of proteindifferences between fresh and stored PLTs. Even with opti-mized storage conditions, we might expect more PLT pro-teins to be altered after 7 days on an agitator. Overall, only93 proteins were identified by gel-based methods, whichis comparable to other studies when redundant proteinsare removed.3 The reliability of two-dimensional (2D) gelshas been improved for quantitative purposes with theadvent of DIGE, where samples are differentially labeledwith fluorescent dyes (e.g., Cy3, Cy5) and simultaneouslyanalyzed in a single gel. DIGE is a major advance in quan-titative gel-based proteomics; however, it still suffers fromthe limitations of any gel-based system including gel-to-gel reproducibility. Despite better standardization of gels

and robotics to pick and digest protein spots, suchprotein-centric techniques remain cumbersome and noteasily applied to large-volume screening.

Other quantitative proteomic approaches are beingused more frequently by investigators wishing to compareprotein expression levels between samples. Thesemethods vary in their ability to simultaneously identifyand quantify peptides. In addition to DIGE, Thon andcoworkers used two other techniques including ICAT andiTRAQ. Based on the PLT storage data, it appears thatiTRAQ is one of the most promising technologies, as sug-gested by others who have compared these methods.4

ICAT reagents, which were commercially available beforethe iTRAQ variety, have been successfully utilized insimilar settings. These reagents have limitations thathinder their resolving power when dealing with complexsamples, but several of the major shortcomings of ICAThave been addressed with iTRAQ reagents. Both ICAT andiTRAQ do require sophisticated and expensive instru-ments at this time, along with specialized analysis soft-ware that facilitates quantifying the differentially labeledpeptides.

To comprehensively study a proteome, it iswell accepted that complementary techniques must beutilized as emphasized by Thon’s group. The choice oftechniques will depend on the ultimate goals of thestudy. The earliest “fishing” stages of proteomic experi-ments determine the universe of proteins that might bealtered by a process, in this case, PLT storage. This strat-egy will produce a list of potential “biomarkers” thatmight be useful in judging PLT quality, along with anumber of misleading proteins. We must remain cogni-zant that the complete makeup of the PLT proteome hasnot been elucidated. Since PLTs can absorb proteins, it isdifficult to determine if PLT proteins are derived frommegakaryocytes or are taken up from the blood. Onehelpful piece of information is the PLT genome since thePLT RNA composition (or PLT “transcriptome”) mayprovide clues as to which proteins might be intrinsic toPLTs.5 Promising biomarkers must be confirmed by othertechniques such as the one selected by the authors,namely, immunoblotting. As in most genomic or pro-teomic studies, investigators are often limited to verify-ing proteins for which antibodies are available, andmany of these antibodies are directed against commonproteins that are highly abundant in cells. Moreover,immunoblotting only substantiates that a particularprotein is present—it does not necessarily prove that theprotein is functional.TRANSFUSION 2008;48:408-410.

408 TRANSFUSION Volume 48, March 2008

As evidenced by Thon’s 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, butThon’s 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 be more capableof identifying hydrophobic membrane proteins. Otherevents on the horizon that may facilitate PLT proteomicefforts include the development of: 1) remarkably sensi-tive mass 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 sophisticated“hybrid” 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 spectrometers—further progress will benefit all.

Numerous tests have been proposed to monitor forPLT storage–related effects. The most 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 PLT monitoring. 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 of mechanisms. 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 or may 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. Thisvalue—which is too low for a genomics experiment wherethousands of genes are tested—may be appropriate fora proteomics experiment. Unfortunately, standards forprotein identification “cutoffs” have not been defined.

EDITORIAL

Volume 48, March 2008 TRANSFUSION 409

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 Pathology

West Virginia UniversityHealth Sciences Center

Morgantown, WVe-mail: pperrotta@hsc.wvu.edu

REFERENCES

1. Downard KM. Ions of the interactome: the role of MS in

the study of protein interactions in proteomics and struc-

tural biology. Proteomics 2006;6:5374-84.

2. Thon JN, Schubert P, Duguay M, Serrano K, Lin S, Kast J,

Devine DV. Comprehensive proteomic analysis of protein

changes during platelet storage requires complementary

proteomic approaches Transfusion 2008;48:420-31.

3. Marcus K, Immler D, Sternberger J, Meyer HE. Identifica-

tion of platelet proteins separated by two-dimensional gel

electrophoresis and analyzed by matrix assisted laser

desorption/ionization-time of flight-mass spectrometry

and detection of tyrosine-phosphorylated proteins. Elec-

trophoresis 2000;21:2622-36.

4. Wu WW, Wang G, Baek SJ, Shen RF. Comparative study of

three proteomic quantitative methods, DIGE, cICAT, and

iTRAQ, using 2D gel- or LC-MALDI TOF/TOF. J Proteome

Res 2006;5:651-8.

5. Gnatenko DV, Dunn JJ, McCorkle SR, Weissmann D, Per-

rotta PL, Bahou WF. Transcript profiling of human plate-

lets using microarray and serial analysis of gene

expression. Blood 2003;101:2285-93.

6. Martens L, Van Damme P, Van Damme J, Staes A, Timmer-

man E, Ghesquière B, Thomas GR, Vandekerckhove J,

Gevaert K. The human platelet proteome mapped by

peptide-centric proteomics: a functional protein profile.

Proteomics 2005;5:3193-204.

7. Palagi PM, Hernandez P, Walther D, Appel RD. Proteome

informatics I: bioinformatics tools for processing experi-

mental data. Proteomics 2006;6:5435-44.

8. Snyder EL, Dunn BE, Giometti CS, Napychank PA, Tandon

NN, Ferri PM, Hofmann JP. Protein changes occurring

during storage of platelet concentrates. A two-dimensional

gel electrophoretic analysis. Transfusion 1987;27:335-41.

9. Reddy KS, Perrotta PL. Proteomics in transfusion medicine.

Transfusion 2004;44:601-4.

EDITORIAL

410 TRANSFUSION Volume 48, March 2008