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Combining results from this and other specialized protein/mRNA analysis methods including MS, immunohistology, and RNA-seq may be the way forward to find biomarkers for Precision Medicine

Combining results from this and other specialized protein/mRNA analysis methods including MS, immunohistology, and RNA-seq may be the way forward to find biomarkers for Precision Medicine. Materials and methods A431 cell lysate containing pTyr-EGFR Two different lots of pTyr-EGFR positive control sample (pE) were purchased from Exalpha (Shirley, MA, Cat # X1003, A431 cell lysates stimulated by EGF, lots 10852 and 13639), referred to as lots 1 and 2. and pY corresponds to phosphorylated tyrosine residue. Fragmentation of the precursor ions in MSMS produced a set of product ions that correspond to the unphosphorylated (top) and phosphorylated (bottom) peptide. The tyrosine residue that is phosphorylated is usually Y1096, while Y1092 is not.(TIF) pone.0234645.s004.TIF (127K) GUID:?750B47B6-4C83-424C-80B1-986BC9055151 S5 Fig: MS spectrum of the precursor ion (inbox) with m/z of 434.26 (2+) that corresponds to phosphorylated peptide ASpYYRK. Note that pY corresponds to phosphorylated tyrosine residue. Fragmentation of the precursor ion in MSMS produced a set of product ions that correspond to the phosphorylated (bottom) peptide. The tyrosine residue that is phosphorylated is usually Y1282, while Y1283 is not.(TIF) pone.0234645.s005.TIF (162K) GUID:?12EA460A-3F8D-4F79-AA43-2C5BBA6B3A12 S6 Fig: MS spectrum of the precursor ion (inbox) with m/z of 1038.61 (2+) that corresponds to phosphorylated peptide TSTIMTDpYNPNYC(#)FAGK Note that C(#) represents cysteine modified by acrylamide (propionamide) and pY corresponds to phosphorylated tyrosine residue. Fragmentation of the precursor ion in MSMS produced a set of product ions that correspond to the phosphorylated peptide. The tyrosine residue that is phosphorylated is usually Y1092, while Y1096 is not.(TIF) pone.0234645.s006.TIF (294K) GUID:?04A78A1D-BB07-4CA0-9D1D-E3F588B0D0CC S1 Table: Band density values and pE/pA density ratios for Run 1 (pE Lot 1) and Run 2 (pE Lot 2) shown in Fig 5. The pE/pA ratios were calculated using the 1 ng pA band density (n = 2 lanes) on the same gel. In Run 1, this value was 1000 for the 3 min 1787 for the 10 min. For Run 2, the value was 1152 for the 3 min and 2110 for the 10 min. Average values contain SD; CV, coefficient of variation = SD/mean *100.(DOCX) pone.0234645.s007.docx (77K) GUID:?C39C8986-1EF7-4507-9F1A-0CEE074FB073 S1 Dataset: Natural data from 1D and 2D western blots underlying all findings. (XLSX) pone.0234645.s008.xlsx (20K) GUID:?A729ABA4-35E6-4FC2-88CD-BD77DD2A94C9 S1 Raw images: Original images behind all figures and data analysis. (PDF) pone.0234645.s009.pdf (2.0M) GUID:?927566C4-8868-4701-90E9-DADBEC4BC9FB Data Availability StatementData may be found within the paper and supporting files. Abstract Protein tyrosine phosphorylation is key to activation of receptor tyrosine kinases (RTK) that drive development of some cancers. One challenge of RTK-targeted therapy is usually identification of those tumors that express RO462005 non-mutated but activated RTKs. Phosphotyrosine (pTyr) RTK levels should be more predictive of the latter than expressed total protein. Western blotting (WB) with a pTyr antibody and enhanced chemiluminescence (ECL) detection is sufficiently sensitive to detect pTyr-RTKs in human tumor homogenates. Presentation of results by comparing WB images, however, is wanting. Here we describe the preparation of a new pTyr-protein standard, pTyr-ALK48-SB (pA), derived from a commercial anaplastic lymphoma kinase (ALK) recombinant fragment, and its use to quantify pTyr-epidermal growth RO462005 factor receptor (pTyr-EGFR) in commercial A431 cell lysates. Linearity of one-dimensional (1D) WB plots of pA band density versus load as well as its lower level of detection (0.1 ng, 2 fmole) were determined for standardized conditions. Adding pA to two lots of A431 cell lysates with high and low pTyr-EGFR allowed normalization and quantification of the latter by expressing results as density ratios for both 1D and 2D WB. This approach is usually semi-quantitative because unknown RTKs may be outside the linear range of detection. Semiquantitative ratios are an improvement over comparisons of images without a RO462005 reference standard and facilitate comparisons between samples. Introduction Receptor tyrosine kinases (RTK) such as epidermal growth factor receptor (EGFR) are large, transmembrane proteins that function in signal transduction. Binding of a serum ligand (EGF for example) to an extracellular protein domain triggers protein dimerization and subsequent trans-phosphorylation of multiple tyrosine residues on intracellular kinase domains. The RTK phosphotyrosines (pTyr) plus adjacent amino acids become RO462005 docking sites for matching Src homology 2 domains on cytosolic proteins. The latter in turn interact to cause cell growth and differentiation. Tyrosine phosphorylation is the key event leading to RTK activity, not protein expression per se. Aberrant pTyr-RTK activity sometimes drives cancer growth [1, 2]. Preliminary results in our laboratory suggested that CORIN standardized 1- and 2-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D and 2D SDS PAGE,.