ICPL
In-depth analysis of proteomes is challenged by the extreme dynamic range of protein abundance and vast complexity. To enable the quantitative analysis of even low abundant proteins, it is indispensable to reduce complexity on the level of proteins by several fractionation steps. To compensate for these time consuming steps and to avoid non reproducible loss of protein species isotope labeling with ICPL Quadruplex is the method of choice to achieve confident results.
Since ICPL is based on stable isotope tagging at the free amino groups of intact proteins, it is applicable to any protein sample, including extracts from tissues or body fluids. After labeling of up to four different proteome states the samples can be combined and the complexity reduced by any separation method currently employed in protein chemistry. After enzymatic cleavage of the protein fractions the ratios of peptides in the different proteome states can be calculated by simple MS1 based mass spectrometric analyses. Only peptides that exhibit regulations in the different proteome states are further investigated for identification by tandem-mass spectrometry. The quantification of multiplexed ICPL experiments is greatly facilitated by the recently published ICPLQuant software, which was especially designed to cover the whole ICPL workflow. The method shows highly accurate and reproducible quantification of proteins, yields high sequence coverage and is indispensable for the comprehensive detection of post-translational modifications and protein isoforms.
To allow for the detection of proteins, which are present only in one sample and therefore would appear as single peptide, additional to the experimental data set a reference data set is prepared.
Experimental sample: Four protein mixtures obtained from four distinct cell states, tissues or body fluids are individually reduced and alkylated to denature the proteins and to ensure easier access to free amino groups. Subsequently proteins are labeled each with one of the four ICPL reagents (ICPL0, ICPL4, ICPL6, ICPL10). After combining all mixtures, any separation method can be adopted to reduce the complexity of the sample on the protein level and, after digestion, on the peptide level. Quantification and identification is done by high throughput MS. Since peptides with identical amino acid sequence derived from the four differentially labeled protein samples differ in mass, they appear as quadruplets in the acquired MS-spectra. From the ratios of the ion intensities of these sister peptide pairs, the relative abundance of their parent proteins in the original samples can be determined. As an integral part of the ICPL strategy the use of a reference sample is highly recommended.
The reference sample is prepared by combining an equal aliquot of all four proteome samples to be analysed. This combined sample is then splitted into 4 equal portions which are treated like the experimental samples, i.e reduction, alkylation, ICPL labelling, protein fractionation and analysis. After enzymatic cleavage of the protein factions and mass spectrometry, each lysine containing peptide will appear as an equi-intense quadruplet. These quadruplets can be stored in a database and by comparison with the data of the experimental samples enable also the identification of incomplete quadruplets (i.e. existence of a peptide in only 1 or 2 out of the four experimental proteomics states). The MS data of the experimental sample and/or the reference sample should be submitted to ICPLQuant, a software especially designed for the ICPL workflow. This enables a rapid and highly automated quantification of the complex ICPL proteomic experiments.
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