Dataset Information

Identification of LMTK3 protein by LC/MS/MS Analysis of 1D SDS PAGE Gel Bands

ABSTRACT: Identification of LMTK3 by LC/MS/MS. A plasmid encoding for full-length LMTK3 was overexpressed in MCF-7 breast cancer cells. Following antibody detection by WB, two bands of interest were seen at around 200 kDa and 150 kDa on the blotting membrane. Colloidal Coomassie (CCBB) staining was performed on a separate gel, to which the Western blot was aligned with the gel markers and a region of gel corresponding to the bands of interest were excised, trypsin digested and analysed by LC/MS/MS.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

DISEASE(S): Breast Cancer

SUBMITTER: Georgios Giamas

LAB HEAD: Georgios Giamas

PROVIDER: PXD001004 | Pride | 2022-02-22

REPOSITORIES: Pride

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Publications

The kinase LMTK3 promotes invasion in breast cancer through GRB2-mediated induction of integrin β₁.

Xu Yichen Y Zhang Hua H Lit Lei C LC Grothey Arnhild A Athanasiadou Maria M Kiritsi Marianna M Lombardo Ylenia Y Frampton Adam E AE Green Andrew R AR Ellis Ian O IO Ali Simak S Lenz Heinz-Josef HJ Thanou Maya M Stebbing Justin J Giamas Georgios G

Science signaling 20140617 330

Lemur tyrosine kinase 3 (LMTK3) is associated with cell proliferation and endocrine resistance in breast cancer. We found that, in cultured breast cancer cell lines, LMTK3 promotes the development of a metastatic phenotype by inducing the expression of genes encoding integrin subunits. Invasive behavior in various breast cancer cell lines positively correlated with the abundance of LMTK3. Overexpression of LMTK3 in a breast cancer cell line with low endogenous LMTK3 abundance promoted actin cyto ...[more]

PMID: 24939894

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Project description:Proteomic analysis by combining PAGE separation, gel slicing and slice-by-slice LC-MS/MS has been frequently reported in recent years. Since the MS analysis would provide identities and quantities of the proteins along the whole lane, visualization by dye staining could be skipped to save time and labor and also in some reports assumed to improve MS identification and sensitivity. In this work, we examined the effect of CBB R-250 staining on the performance of the method and the results showed actually better results were obtained with CBB staining than without. A primary examination was firstly performed with gel bands of purified proteins, in which eight protein bands were excised, each from both the CBB-stained and unstained gel parts, and then analyzed by in-gel digestion and quantitative LC-MS/MS. Almost all the proteins were detected with higher sequence coverages and quantities from the stained gel bands than from the unstained. Then a proteomic sample of rat heart soluble proteins was examined for the complete workflow. The sample was firstly separated by nondenaturing PAGE and the gel was divided to two halves, with one CBB-stained and the other unstained. Laboratory-made tools were used to simultaneously cut duplicate lanes from each gel half and then slice each lane into about 39 pieces of the same size (1.1 mm 椋?1.1 mm 椋?1 mm thick). All the gel square pieces were analyzed in standardized procedures of in-gel digestion, peptide extraction and label-free quantitative LC-MS/MS. The results showed an average of 1434 proteins were detected in CBB-stained lanes, 40% higher than the 1013 in unstained lanes. When proteins detected in both conditions were compared, most of them were detected in higher quantities and in more gel squares with CBB staining. The comparison was also performed for SDS-PAGE and similarly advantageous results were obtained from the CBB-stained lanes, in both the detected numbers of proteins and peptides and the detected quantities and square numbers of individual proteins. The data also showed the proteins with lower molecular masses (e.g. < 30 kDa) were more benefited by the staining, probably because the dye binding helped to retain the proteins in gel matrix. In short, though dye staining is no longer a requisite when PAGE separation is followed by whole-gel LC-MS/MS analysis, CBB staining is still recommended for the better detection in proteomic analysis. All the raw and search files of the LC-MS/MS analysis, for (8*4=) 32 gel squares of HMW and LMW markers and (39*4+41*4=) 320 squares of rat heart soluble proteins (totally 704 files), as well as the gel patterns (2 files) and the summaries of the protein-level search results (3 files), are deposited in this project.

Dataset Information

Identification of LMTK3 protein by LC/MS/MS Analysis of 1D SDS PAGE Gel Bands

Publications

The kinase LMTK3 promotes invasion in breast cancer through GRB2-mediated induction of integrin β₁.

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