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Haematological Malignancies
New Challenges this technique could modify the protein ratio, and in doing so impede
Proteomics has evolved to be an accurate strategy that provides proteome comparison between different clinical or biological stages.
informative data on disease pathogeny. Three new specific challenges for Preliminary studies on cerebrospinal fluid have shown that quantitative
proteomics in future clinical are: information and the relative ratio between different samples were
maintained after equalisation.
• mining the proteome;
• improving MS-based quantitative proteomics; and Improving Mass Spectrometry-based
• interpreting the protein language using proteomics. Quantitative Proteomics
MS is not inherently quantitative and allows for, at best, a semi-
Mining the Proteome quantitative evaluation of protein based on the comparison of the
One major problem with proteomic studies, regardless of the peptide peak intensity between two different source samples.
experimental approach, is the wide concentration of range in the Quantification of proteins in parallel with their identification is necessary
biological samples. This is particularly true in body fluids, where the to produce proteomic clinically relevant data.
concentration range of proteins spans 10 or even 12 orders of
magnitude. The most abundant protein, human serum albumin, The ‘stable isotope labelling’ method remains the core technology for
constitutes over 50% of the total protein content in plasma. This is the proteomic quantification. Interest in chemical labelling for quantitative
reason why most proteomics studies publish non-specific biomarkers, MS was first shown in 1999,
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when the isotope-coded affinity tag (ICAT)
which are more likely to be linked to the inflammatory response than to technology, based on a cysteine-specific tagging of intact proteins, was
the pathogenic process. Given this, there is a clear need to improve the introduced. Alternative chemical labelling methods, such as the isobaric
pre-analytical preparation of samples in order to detect low-abundance tag (iTRAQ), were later developed and introduced the stable isotope of
proteins and identify specific plasma or serum cancer biomarkers.
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proteolytic peptides.
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A limitation of the isotope method, despite the
relative complexity of the isotope, is the low number of samples that can
To overcome the limited range of MS detectors, several candidate be quantified at the same time (maximum of eight). An example of such
technologies have been proposed. Depletion of the most abundant a strategy is provided in a study that employed the isobaric tag peptide
proteins, such as albumin, transferrin and immunoglobulins, by labelling (iTRAQ) coupled with nano-LC tandem MS to analyse the effect
immobilised dyes or immuno-affinity may be the most intuitive strategies. of imatinib on the CML cell proteome. Large populations of proteins and
While this may give rise to interesting results, it may also deplete the novel consequences of the action of the imatinib were identified. In
peptides (or low-molecular weight proteins) at the same time. These particular, DEAD-box protein-3, Hsp105 and peroxiredoxin-3 were
peptides are the best candidates for bio-marking in oncology. identified as potential biomarkers for a response to imatinib.
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Two strategies focus on the interest and effort of a proteomics platform. Due to continuing technical improvements, new generations of MS offer
The first performs an extensive fractionation (anion exchange improved sensitivity and a higher sequencing speed, allowing for the
fractionation, reversed-phase chromatography) before analysis of label-free quantitative analysis of samples. Samples are separately
individual fractions.
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The reduction in complexity of individual fractions analysed by nanoLC-MS/MS and the peak intensity of peptides is
allows for the discovery of low-abundance proteins further to LC-MS/MS. compared, providing information on quantitative differences. New
Selection of a subset of proteins by affinity chromatography using, for generations of FT mass spectrometers offer improved sensitivity and
example, multiple lectins to capture glycoproteins, allows a decrease in significantly higher sequencing speeds, typically enabling the identification
the sample complexity. One pitfall of this strategy is that it relies on the of several thousands of proteins.
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Linear ion trap (LTQ) combined with a
persistence of major proteins in some fractions that do not modify the new mass analyser, the orbitrap, can achieve very high resolutions and
concentration range. mass accuracy. It is very likely that by maximising the number of proteins
identified during a nanoLC-MS/MS run and allowing the comparison of
The second strategy consists on enriching samples in low-abundance large numbers of patients, these tests will be frequently performed,
proteins while decreasing the concentration of the most abundant. The despite the significant investment needed to achieve this.
goal is to obtain, through an equalisation-bead-technology and using a
library of combinatorial ligands: ‘fixed-on’ beads.
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Highly specific affinity Interpreting Protein Language Using Proteomics
interactions between proteins and ligands ensure the binding of the One major element of the regulation of proteome function is the
greatest number and variety of proteins. The result is the concentration of consequences of post-translational modification (PTM), which define
rare species and the dilution of highly abundant species that narrow the the structural plasticity of proteins. PTMs such as phosphorylation,
concentration range of proteins present in the sample. Montasarrat and glycosylation and acylation increase molecular heterogeneity – more
colleagues applied a highly purified fraction, obtained with equalisation than 200 types of PTM have been characterised – and represent
bead technology (nanoLC-MSmS) to a LTQ-Orbitrap MS. They could significant markers of the gene diversity playing a fundamental role in
confidently identify as many as 1,578 proteins in the cytoplasmic fraction cell biology. Constitutive phosphorylation, due to mutations in
of erythrocyte, which allowed a deep exploration of the classic red blood tyrosine kinase, is present in a significant proportion of haemopathy
cell (RBC) pathway and the identification of unexpected minor proteins.
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and represents critical events in the pathogenesis of MPD-Phi-. PTM is
Platelet proteome was also significantly explored.
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recognised as an important target for molecular medicine.
The main potential pitfall of this method relates to its impact on relative MS allows for the site-specific assignment of PTM to resolve individual
concentrations of proteins. Modification of concentrations of proteins by amino acids in proteins.
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The interest in an MS strategy as a functional
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