Final Report Summary - METAG (A new 2D-LC approach for quantitative proteomics using MeCAT labeling and multidimensional chromatography coupled to ultra-high-resolution FT-MS-MS and ICP-MS)
The analysis of a proteome means the analysis of all proteins in a cell, a tissue or another biological entity at a given time. Qualitative and quantitative determinations are both fundamental to characterize the state of the biological system. After the great progress achieved in qualitative proteomics, the quantitative dimension emerge as the new challenge.
In this project, MeCAT (metal-coded affinity tags) (Figure 1) reagents have been used to labeled proteins allowing the quantification of the labeled species by elemental mass spectrometry techniques such as ICP-MS (inductively coupled plasma mass spectrometry). The introduction of ICP-MS for quantification tasks is the most novel aspect of the project and present important advantages as high sensitivity, structure-independent signal, wide dynamic range and multi-elemental capability. Thus, minor proteins together with abundant proteins can be quantified employing simple external calibrations or even using more sophisticated quantification techniques as isotope dilution analysis (IDA).
Using the described strategy, three different approaches have been developed for the absolute quantification of proteins. The first one consisted of the digestion and labeling of the proteins with a MeCAT reagent followed by LC(RP)-ICP-MS analysis. Some standard labeled peptides were used as standards to build up a calibration curve. The novelty of this approach is the measurement of the calibration points after the chromatographic separation, compensating possible losses of sample on the column. Samples of low or moderate complexity were quantified successfully.
An off-line double chromatographic separation was designed for the separation of complex samples. In this case, the standard labeled peptides were used as internal standards to estimate the losses of the specific peptides along the procedure. In this approach also was tested the multiplex capability of the MeCAT reagent. Several proteins were labeled with different lanthanides in the MeCAT reagent and finally were quantified in the same run thanks to the multi-elemental detection of the ICP-MS.
Finally, a protein level approach was developed using gel electrophoresis as separation technique. IDA was used instead of conventional external calibration to improve the accuracy and speed in the quantification. For this purpose, standard labeled proteins were prepared with enriched MeCAT labels while the sample was labeled with the natural form of the same reagent. Several standard proteins as well as blood serum proteins in real samples were quantified successfully. Moreover, excellent results were obtained measuring the proteins both by mineralization of the electrophoretic spot or by direct laser ablation (LA) of the gel.
In all the described methodologies, ESI-MS/MS (electrospray tandem mass spectrometry) was used to identify the detected peptides; therefore, it can be conclude that the combination of elemental and molecular mass spectrometry is a powerful tool for quantitative proteomics. In addition, it has been demonstrated that MeCAT reagents can be employed for quantification purposes, also in multiplex approaches, not only at peptide level but also analyzing intact proteins. Finally, must be highlighted the potential of IDA as quantification technique and the fast and accurate determinations performed by LA-ICP-MS.
This project has developed new and sensitive analytical methodologies for the quantification of proteins. The proposed strategies based on MeCAT labeling and ICP-MS detection may help to advance on currently hot topics like the investigation of the cell cycle, the development of drugs with specific effects or the quest for significant diseases bio-markers.
In this project, MeCAT (metal-coded affinity tags) (Figure 1) reagents have been used to labeled proteins allowing the quantification of the labeled species by elemental mass spectrometry techniques such as ICP-MS (inductively coupled plasma mass spectrometry). The introduction of ICP-MS for quantification tasks is the most novel aspect of the project and present important advantages as high sensitivity, structure-independent signal, wide dynamic range and multi-elemental capability. Thus, minor proteins together with abundant proteins can be quantified employing simple external calibrations or even using more sophisticated quantification techniques as isotope dilution analysis (IDA).
Using the described strategy, three different approaches have been developed for the absolute quantification of proteins. The first one consisted of the digestion and labeling of the proteins with a MeCAT reagent followed by LC(RP)-ICP-MS analysis. Some standard labeled peptides were used as standards to build up a calibration curve. The novelty of this approach is the measurement of the calibration points after the chromatographic separation, compensating possible losses of sample on the column. Samples of low or moderate complexity were quantified successfully.
An off-line double chromatographic separation was designed for the separation of complex samples. In this case, the standard labeled peptides were used as internal standards to estimate the losses of the specific peptides along the procedure. In this approach also was tested the multiplex capability of the MeCAT reagent. Several proteins were labeled with different lanthanides in the MeCAT reagent and finally were quantified in the same run thanks to the multi-elemental detection of the ICP-MS.
Finally, a protein level approach was developed using gel electrophoresis as separation technique. IDA was used instead of conventional external calibration to improve the accuracy and speed in the quantification. For this purpose, standard labeled proteins were prepared with enriched MeCAT labels while the sample was labeled with the natural form of the same reagent. Several standard proteins as well as blood serum proteins in real samples were quantified successfully. Moreover, excellent results were obtained measuring the proteins both by mineralization of the electrophoretic spot or by direct laser ablation (LA) of the gel.
In all the described methodologies, ESI-MS/MS (electrospray tandem mass spectrometry) was used to identify the detected peptides; therefore, it can be conclude that the combination of elemental and molecular mass spectrometry is a powerful tool for quantitative proteomics. In addition, it has been demonstrated that MeCAT reagents can be employed for quantification purposes, also in multiplex approaches, not only at peptide level but also analyzing intact proteins. Finally, must be highlighted the potential of IDA as quantification technique and the fast and accurate determinations performed by LA-ICP-MS.
This project has developed new and sensitive analytical methodologies for the quantification of proteins. The proposed strategies based on MeCAT labeling and ICP-MS detection may help to advance on currently hot topics like the investigation of the cell cycle, the development of drugs with specific effects or the quest for significant diseases bio-markers.