**7. Proteomics in acute leukemia**

*Proteoforms - Concept and Applications in Medical Sciences*

**6.1 MS-based**

**6.2 Antibody-based**

cells (approximately 3 × 105

on the number of antibodies printed on the slide.

provide useful information about both protein abundance and activity, as regulated by the PTM, the protein-protein and protein-DNA interactions. Nowadays, two of the most commonly used high-throughput techniques to study the proteome in leukemia are mass-spectrometry (MS)-based techniques and antibody-based techniques.

MS is a high-throughput technique uses the formation of ions (charged fragments) from the protein analyte to distinguish between proteoforms. Those ions can be sorted and measured using electrical and/or magnetic fields based on their mass-to-charge ratio (m/z), and identification of the protein follows based on the abundance of those m/z-fragments [40]. Globally, proteins can be ionized with two distinct methods: matrix assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI). In MALDI the protein sample is mixed with an energy absorbing matrix. Irradiation of this matrix causes vaporization of the matrix together with the sample, resulting in the formation of ions [41]. ESI creates ions using electrospray to dissolve the protein lysate, by applying high-voltage to the dissolvent to create an aerosol of small charged fragments. When a protein sample is highly complex, samples may require separation prior to MS analysis using 1D or 2D gel electrophoresis, high-pressure liquid chromatography (LC-MS), or gas chromatography (GC-MS) to maximize the sensitivity. Because proteoforms are derived from a single gene, they often contain homologous sequence regions, and because of the digestion step, information about the relationship between amino acid sequence and the PTM often lacks, this significantly complicates the process of identifying proteoforms. Several overviews have been published that discuss recent technological developments of MS to enable analysis distinct proteoforms [42–44].

Another high-throughput approach is the protein microarray (PMA), of which two different types exist: forward phase protein arrays (FPPA) and reverse phase protein arrays (RPPA). Given that antibodies can be raised to specifically recognize sequence variations or PTM, they enable measurement of selected proteoforms. In FPPA, protein antibodies are immobilized on an array in known positions, and samples are then printed on the array. If a particular proteoform is present in the sample, the proteoform binds to the antibody and after exposure to a secondary antibody, the abundance can be measured. Each slide is incubated with a single protein sample, but multiple proteins can be measured simultaneously depending

The "reverse" version of the FPPA is the RPPA methodology. In RPPA, samples are first printed on the array, and subsequently each slide is stained with a single protein antibody, followed by a secondary antibody to amplify the signal. The downsides of RPPA are that all samples must be printed at the same time to avoid methodological barriers due to printing irregularities between batches, and that RPPA can only be used to detect proteins for which a strictly validated antibody is available. As there is no separation of the proteins according to molecular weight, it is crucial that antibodies are proven to be highly specific, selective and reproducible. Plus, RPPA is biased to proteins and isoforms for which a strictly validated antibody is available. On the other hand, RPPA requires only a small number of

suitable for retrospective clinical applications. As it in addition analyzes all samples

at once, it allows a direct comparison of protein abundance across samples.

cells to test 400 different antibodies), making it highly

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