Abstract

   The activation of signaling cascades in response to extracellular and
   intracellular stimuli to control cell growth, proliferation and
   survival, is orchestrated by protein kinases via phosphorylation. A
   critical issue is the study of the mechanisms of cancer cells for the
   development of more effective drugs. With the application of the new
   proteomic technologies, together with the advancement in the sequencing
   of the human proteome, patients will therefore be benefited by the
   discovery of novel therapeutic and/or diagnostic protein targets.
   Furthermore, the advances in proteomic approaches and the Human
   Proteome Organization (HUPO) have opened a new door which is helpful in
   the identification of patients at risk and towards improving current
   therapies. Modification of the signaling-networks via mutations or
   abnormal protein expression underlies the cause or consequence of many
   diseases including cancer. Resulting data is used to reveal connections
   between genes proteins and compounds and the related molecular pathways
   for underlining disease states. As a delegate of HUPO, for human
   proteome on children assays and studies, we, at Hospital Universitario
   Niño Jesús, are seeking to support the human proteome in this context.
   Clinical goals have to be clearly established and proteomics experts
   have to set up the appropriate proteomic strategy, which coupled to
   bioinformatics will make it possible to achieve new therapies for
   patients with poor prognosis. We envision to combine our up-coming data
   to the HUPO organization in order to support international efforts to
   advance the cure of cancer disease.

   Keywords: Diagnosis, Oncology, Proteomics, Treatment

Background

   Proteomics is a powerful tool in biomarker discovery and mechanism
   understanding. Proteomics is the next generation following genomics.
   Using proteomics, researchers can efficiently perform large-scale
   screening to achieve valuable information [[30]1].

   The Human Proteome Organization (HUPO) main goal is to serve the Public
   Health Service in an international manner via collaborators from the
   best expertise and well-known excellence academics. In this context, we
   would like to point out the important contribution from Spanish
   Proteomic Society (SeProt), European Proteomic Society (EUPA) and HUPO
   to obtain the sequence of the human proteome. We aim to contribute with
   our future data from Hospital Niño Jesus using body fluids (such as
   bone marrow, peripheral blood) from children suffering cancer, and by
   collaboration with several international hospitals and research centers
   of prestige for this important labor, which will benefit children with
   poor prognoses as at present there is no correct therapy for all types
   of pediatric cancers [[31]2–[32]5].

   As different phosphosites in a protein trigger either protein
   activation or inactivation; phosphosites can be used for
   quantification. The high number which can be identified as altered
   phosphoproteins in a clinical study, implies that many can also be
   reported with key roles in tumor progression and/or drug resistance.
   Indeed, when obtaining phosphoproteomics data in clinical research,
   vast knowledge of drug resistance appear, and thus, new insights are
   offered for future drug candidates. Several articles show that
   proteomic analysis is a powerful tool for profiling the phosphorylation
   patterns and may help to better understand drug resistance
   [[33]6–[34]11]. We aim to show some proteomic and bioinformatics tools
   which are useful for clinical research and which contribute towards
   accurate diagnoses and improve therapies in order to benefit the
   patients.

Findings

Phosphoproteomics for deciphering drug resistance and new therapies related
to phosphorylation pathways in cancer

   Our aim is to outline as basic ideas and tools of proteomics, how
   evolution is aiming to reach clinical advances. Amino acids
   site-specific phosphorylation assignments on thousands of proteins in a
   single experiment are possible. The combination of different
   proteomic-MS strategies is already being carried out to characterize
   signaling pathways that govern oncogenesis and also to unravel targets
   of kinase inhibitors, difficult to characterize because of spatial and
   temporal cellular events. It is, therefore, helpful for understanding
   cell pathways and facilitating drug discovery [[35]1].

Sample preparation

   The sample preparation step is the key to successful
   phosphoproteomic-analysis. These include: (a) be snap-frozen; (b)
   include treatments with phosphatase inhibitors to avoid modifying
   phosphopeptides during sample work-up; and (c) avoid salts and
   detergents, which interfere with subsequent analyses. Using Immobilized
   metal ion affinity chromatography (IMAC), titanium dioxide metal-based
   chromatography (TiO[2]), zirconium dioxide (ZrO[2]) and sequential
   elution from IMAC (SIMAC), the negatively charged phosphopeptides are
   purified by their affinity to positively charged metal ions [[36]1,
   [37]8].

   During IMAC and TiO[2] operations, simple and complex samples
   containing phosphopeptides and non-phosphorylated peptides are
   dissolved in an acidic solution to reduce the non-specific binding of
   acidic peptides, and to stimulate the electrostatic interactions
   between the negatively charged peptides, mainly phosphopeptides, and
   the metal ions. IMAC mainly elutes multiple-phosphopeptides while
   TiO[2]chiefly elutes mono-phosphopeptides. Both resins have the
   drawback of binding acidic non-phosphorylated peptides (negatively
   charged peptides), as peptides containing acidic amino acid residues
   (e.g. glutamic acid and aspartic acid), can also bind to the metal
   ions. This drawback on IMAC (Fe^3+) is circumvented via converting
   acidic amino acid residues to methyl esters and esterification of the
   acidic residues prior to the MS analysis. In addition, higher
   specificity is achieved and yield compared to IMAC (Fe^3+) for the
   selective enrichment of phosphorylated peptides from model proteins
   when using 2,5-dihydroxybenzoic acid (DHB) with TiO[2]. In fact, more
   phosphopeptides are bound to the metal ions and more phosphopeptides
   can be isolated by using ammonium hydroxide as the eluent by use of
   glycolic acid in the loading buffer of TiO[2]. SIMAC is the combination
   of IMAC with TiO[2] protocols. It includes improvements in both resins
   to be coupled in an efficient manner in order to allow enrichment of
   mono-and multiple-phosphorylated peptides in a single experiment.
   Mono-phosphorylated peptides mainly elute from IMAC (Fe^3+) under
   acidic conditions whereas multi-phosphorylated peptides elute at high
   basic pH.

   ZrO[2] is another useful phosphopeptide enrichment prior to MS analysis
   and its principle is based on metal affinity chromatography such as
   IMAC and TiO[2]. The relevant clue related to ZrO[2] is that it permits
   the isolation of single phosphorylated peptides in a more selective
   manner than TiO[2] [[38]12–[39]24]. In addition, purification of
   phosphorylated proteins can be carried out via antibody-purification.
   This methodology is highly efficient when purifying tyrosine
   –phosphorylated proteins, and it can also be coupled to
   phosphoenrichments such as IMAC, TiO[2], ZrO[2] and SIMAC for further
   MS analysis. This is an important advantage as phosphorylation on
   tyrosines is under-represented by MS-assays, thus the use of specific
   antibodies to enrich tyrosine phosphorylated peptides from complex
   samples is of advantage [[40]25].

   When combining the previously mentioned phosphoenrichments with strong
   cation and anion exchange (SCX and SAX) or hydrophilic interaction
   chromatography (HILIC), large-scale phosphoproteomic studies of
   interest can be carried out successfully. During SAX operations, a
   negatively charged analyte is attracted to a positively charged solid
   support, and during SCX operations, a positively charged analyte is
   attracted to a negatively charged solid support. Both techniques were,
   for the first time, successfully coupled to IMAC, achieving greater
   recovery and identification by MS of important phosphorylated peptides
   originating from signalling pathways and membrane proteins
   respectively, therefore making it possible to carry out relevant
   scientific studies following those protocols described in the
   references previously detailed. In addition, today’s scientists use