Bech-Serra, Joan Josep

Canals, Francesc

Colomé, Núria

Monge-Azemar, Marta

Repositorio Institucional de la Universidad de Córdoba

3527(Ï0,&$1Ò0(52)(%5(52 1057KH¿HOGRISURWHRPLFVLQFOXGHVDZLGHQXPEHUof technologies aimed to the analysis of large number of proteins, representing ideally the entire proteome, in the same experiment. In its early stages , mass spectrometry-based proteomics was successfully used to the qualitative characterization of complex mixtures of proteins, leaving the quantitative aspects in a secondary role, essentially because of the lack of suitable technologies for quantitative analysis of such complex mixtures. However, in the last years, a wide number of strategies have been developed LQRUGHUWRJLYHWRWKHSURWHRPLFV¿HOGUREXVWWRROVto obtain reliable quantitative data. Those strategies include both 2D-gel based and non-based methodo-ORJLHV,QWKH¿UVWJURXS'',*(PHWKRGRORJ\LVEDVHGRQWKHODEHOLQJZLWKGLIIHUHQWÀXRURFKURPHVenabling the simultaneous separation of different samples in the same 2D-gel, which allows to over-A comparison of quantitative proteomics methodologies on a differential experiment on test samplesJoan Josep Bech-Serra, Núria Colomé, Marta Monge, Francesc CanalsLaboratori de Proteòmica, Programa de Recerca en Oncologia Mèdica. Institut de Recerca. Hospital Vall d’Hebron, Barcelonacome reproducibility problems inherent to the 2D procedure and internal standardization of spot vo-lume measurements, providing a robust quantitative comparison technique. Alternatively, a number of gel-free quantitative techniques can be used. Some of them are based on non-isobaric isotope labeling, such as ICAT, ICPL or SILAC labeling methodo-logies, the quantitative data being obtained from the from the MS data, by integrating extracted ion chromatograms of the heavy-light peptide pair mas-ses. A second group which includes iTRAQ or TMT reagents is based on isobaric labeling that introduce different reporter fragments in the derivatized pep-tides. Quantitative data is in this case obtained at the MS-MS level, from the relative intensities of the reporter fragment ions. Finally, a third group of label-free methods uses different approaches for quantitative comparison of LC-MS data.curacy has inspired the development of new quan-titative approaches [2]. In particular, label free hy-brid identity/pattern-based approaches have gained SRSXODULW\LQWKHELRPDUNHUGHYHORSPHQW¿HOG>@Such approaches use pattern recognition algorithms to ex post facto assign the identity of LC-MS peaks against databases of peptide sequence, mass, and retention time built from multiple experiments. Here we employ the accurate mass and time (AMT) stra-tegy to identify and quantify peptides/proteins from unfractionated CSF samples using msInspect pla-WIRUP>@2XUDQDO\VLVZRUNÀRZH[WHQGVDQGFRP-ELQHVH[LVWLQJRSHQVRXUFHSODWIRUPVIRU/&í06067UDQV3URWHRPLF3LSHOLQHDQG/&í06PV,QV-pect) data analysis. A peptide accurate mass and LC elution time AMT database was initially generated using MS/MS following extensive multidimensional LC separations to provide the basis for subsequent SHSWLGHLGHQWL¿FDWLRQV2XU&6)$07GDWDEDVHFRQ-tains >2,000 entries from a total number of ~7,500 LGHQWL¿HGSHSWLGHVZKLFKWUDQVODWHVLQWRFRQ¿-GHQW&6)SURWHLQLGHQWL¿FDWLRQV)LJXUH,QFRQ-clusion, hybrid identity/pattern-based approaches are amenable for high throughput quantitative pro-teome analyses in biomarker discovery pipelines.References[1] Zougman, A et al. Integrated Analysis of the Cerebrospinal Fluid Peptidome and Proteome. J Proteome Res 2007; 7: 386-399.[2] Mueller, LK et al. An Assessment of Software Solutions for the Analysis of Mass Spectrometry Based Quantitative Proteomics Data. J Proteome Res 2008; 7: 51-61.[3] Jaffe, J et al. PEPPeR, a Platform for Experi-mental Proteomic Pattern Recognition. Mol Cell Proteomics 2006; 5:1927-1941.[4] May, D et al. A Platform for Accurate Mass and Time Analyses of Mass Spectrometry Data. JProteome Res 2007; 6: 2685-2694.106 3527(Ï0,&$1Ò0(52)(%5(52Figure 1. 6FKHPHRIWKHPHWKRGVFRPSDUHGIRUWKHUHODWLYHTXDQWL¿FDWLRQRIWZRWHVWVDPSOHVIn order to evaluate and compare the perfor-mance of different quantitative proteomics methods, we have prepared a set of two test samples to be compared quantitatively. The samples consist on a matrix of cytoplasmatic E. Coli proteins, of medium complexity (around 100 proteins), to which four standard mammalian proteins have been spiked in different amounts, ranging from the fmol to the pmol level per microgram of total protein.. The ra-tios of the spiked proteins between the two samples have been chosen to range form 1.5:1 to 5:1.These samples have been analyzed by different methodologies (Figure 1): - 2D-DIGE, using four technical replicas of each sample on a 4 2D-gel experiment.- Labeling with ICPL reagents at the protein level, followed by tryptic digestion and analysis by LC-MS. This is the standard procedure for this methodology, but has the drawback that only lysine FRQWDLQLQJSHSWLGHVZKLFKKDYHEHHQPRGL¿HGE\the reagent can be used for quantitation. This results RIWHQLQDODUJHSDUWRIWKHLGHQWL¿HGSURWHLQVODFNLQJquantitative information.- Labeling with ICPL at peptide level. In order to overcome this problem, we have explored an alternative procedure introducing the ICPL labeling step after tryptic digestion of the protein mixtures. This should result in a more comprehensive quan-titative information, since all tryptic peptides will be derivatized at the N-terminus. The weakness of this strategy is that digestion of the two samples has to be run independently, which can introduce technical bias. To minimize the problem of a pos-sible imbalance, both labeling schemes are run in parallel on sample aliquots. This way the balance correction derived from the protein level labeling experiment can be used to normalize the data from the second experiment.ICPL labeled samples have been analyzed in four independent LC-MS runs in order to assess reproducibility.- iTRAQ Labeling. The samples have been analyzed using an 8-plex iTRAQ reagent, using four different reporter masses for each of the samples, and analyzed by LC-MALDI.- Label-free quantitation. Four LC-MS runs of a tryptic digest of each sample have used to quantitati-vely compare the two samples using Progenesis LC-MS software for alignment and statistical analysis of the LC-MS maps.The results of all the quantitative proteomics methods used have been evaluated in terms of accu-racy and reproducibility, and their performance and robustness is discussed.

A comparison of quantitative proteomics methodologies on a differential experiment on test samples

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