Development And Application Of A Novel Method For Multiplex Proteomic Quantitation Based On Metal Element Labeling Coupled With Mass Spectrometry

Quatitative proteomics provide a powerful tool to discover potential biomarkers andsignificant functional proteins. However,the development of methods for accuratequantitation of protein expression in biological samples under different conditions isone of the most challenging area in proteomics. The current approaches based onisotope labeling for multiplex quantitative proteins, for example, isobaric tagging formultiplex relative and absolute protein quantitation (iTRAQ), have disadvantages suchas expensive labeling reagents and costly time of MS/MS quantitation. Therefore it iscurrently hot and competitive area to develope new methods and technique being oflower-cost, robust and accurate merits in proteomics.Recently, metal element labeling has been introduced as a new, alternative methodfor proteomic quantitation. Analogous to isotope labels, metal labels are also nearlychemically equivalent with mass distinguishable tags because of the similar structureand properties of rare earth element. Compared with stable isotope labeling methods,this metal element labeling method has the advantages of much lower cost oflabeling reagents, relatively large isotopic shifts and a lot of rare earth atoms that canbe used for multiplex labeling. Moreover, metal element labeling can be combinedwith molecular mass spectrometry (MALDI-TOF-MS and ESI-MS) and InductivelyCoupled Plasma Mass Spectrometry(ICP-MS)to perform both relative and absoluteprotein quantitation.In this study, for the first time, we established a novel method for multiplex relativeprotein quantitation using amine-reactive metal element chelated tags andMALDI-TOF-MS or ESI-MS, therefore providing an alternative, relatively low-cost,accurate and reliable method for multiplex protein quantitation in proteomics.In the first part of this thesis, four lanthanide metals (Tb, Ho, Tm, Lu) with suitablemass difference and excellent stability were selected as the tags for chelating with the macrocyclic compound of DOTA. The labeling process is a two-step reaction.The first step is the coupling reaction of DOTA-NHS-ester with NH2group ofpeptides. The second step of the labeling procedure is the chelating reaction of themodified peptide with lanthanide metal ions (TbCl3, HoCl3, TmCl3and LuCl3). Weoptimized the reaction conditions by investigating a few factors including pH, buffer,ratio of DOTA-NHS-ester to peptide, reaction temperature, reaction time and ratio ofwater to organic solvent and reaction temperature. Under the optimized conditions,high labeling efficiency of up to99%was achieved by the reagent ofDOTA-NHS-ester reacting with amine of standard synthetic peptides and thenchelating to metals, and for99%of the tryptic peptides from Thermoanaerobactertengcongensis high labeling efficiency was also obtained. The results showed thatthe metal element labeling is a mild, efficient, non-peptide discriminating labelingmethod.In the second part of this thesis, multiplex quantitation of peptides and proteins hasbeen explored by the metal element labeling combined with MALDI-TOF-MS. FourMental element Chelated tags (Tb, Ho, Tm and Lu-MECT) were used for themultiplex protein quantitation. This method shows good reproducibility and accuracy.Good linear correlations (R2=0.99) were achieved for the labeled peptides with theratio ranging from0.2to5. The relative standard deviation (RSD) was below20%for each measured point. In addition, the metal labeled peptides are stable and nometal exchange of the tagged peptides happens under low pH. CID-MS/MS behaviorin MALDI-TOF-MS was also studied. Complete y-series ions and some b-series ionsare observed in MS/MS spectra of the metal labeled peptides, which is significant forthe identification of unknown peptides in a complex sample. Thus multiplex relativequantitation of peptides and proteins using amine-reactive metal chelated tagscoupled with MALDI-TOF-MS holds promising prospect in quantitative proteomics.In the third part of this thesis, the strategy of metal element labeling coupled withESI-MS was also established for multiplex quantitation of peptides and proteins.After guanidination of lysine residues, metal element labeling at the N-terminus ofpeptides was performed, and then the four-plex metal labeled peptide mixtures wereperformed by nano RP-HPLC-ESI-LTQ-FT-MS/MS analysis. Four metal labeledpeptides on reversed phase liquid chromatography coeluted and had almost the sameCID-MS/MS behavior in ESI-LTQ-FT-MS. Compared with the unlabeled peptide, the four metal tagged peptides containing different metals had improved signals ofb-series ions, which could enhance the confidence level for peptide identification.The feasibility for multiplex peptide and protein quantitation of the strategy wasfurther evaluated by using standard peptides and six standard proteins as models.Good linear correlations (R2=0.99) were achieved for the labeled peptides with theratio ranging from0.1to10(Tb/Ho, Tm/Ho, Ho/Tb and Lu/Tb). For the six proteins,the error was about13%, the standard deviation (SD) is up to9%and the relativestandard deviation (RSD) was below20%. The result showed that the method isaccurate, precise and repeatable, indicating it has the potential of being applied tomore complex samples.In the last part of the thesis, the multiplex quantitation method of metal elementlabeling coupled with ESI-MS/MS was applied to the proteome relative quantitationof Thermoanaerobacteria tengcongensis cultured at four different temperatures. Thedata analysis was performed with MaxQuant software and then the quantitationresult was given.13proteins expressed differently as temperaure changing werediscovered from the complex samples with99%confidence level and at least twonon-redundant peptides matched. Among them,8proteins were up-regulated and5proteins were down-regulated with temperature rising. In addinition,7proteinsamong them were found in previous reports:4up-regulated proteins belong toenergy production and conversion protein group and3down-regulated proteins arechaperone protein. Furthermore, others6proteins found expression viaration atdifferent temperatures have not been reported before. This preliminary result provedthe feasibility of metal element labeling combined with ESI-MS in the multiplexprotein quantitation for complex samples.In conclusion, our research provided an alternative strategy to stable isotope labelingfor multiplex proteome relative quantitation. It has expanded the application field ofmetal element labeling techniques in proteomics study.