Chapter 8. Quantitative LC-MS of Proteins

Stöhr, G.; Tebbe, Andreas

doi:10.1039/9781849733144-00104

Cited by 6 publications

(10 citation statements)

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“…To avoid contamination of light amino acids, sub‐culturing was performed using Cell dissociation buffer (Thermo Fisher Scientific) instead of trypsin. Isotopic incorporation was checked using a script in R as previously described 13 after approximately five cell divisions to confirm complete (>95%) labeling. Arginine‐to‐proline conversion was assessed by calculating the percentage of heavy proline (Pro‐6) containing peptides among all identified peptides, and kept at <5%.…”

Section: Methodsmentioning

confidence: 99%

The importance of the ZBED6‐IGF2 axis for metabolic regulation in mouse myoblast cells

et al. 2020

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Section: Methodsmentioning

confidence: 99%

The importance of the ZBED6‐IGF2 axis for metabolic regulation in mouse myoblast cells

et al. 2020

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“…The extent of isotope incorporation was assessed using an R-script as described. 25 Cell lysates were prepared, quantified, subjected to SDS PAGE and in-gel tryptic digest as described previously. 26 At least three biological replicate data sets representative of each KRAS MUTANT versus Parental SW48 were obtained ( n = 4 for KRAS G12D versus Parental).…”

Section: Materials and Methodsmentioning

confidence: 99%

“…To generate light, medium and heavy stable isotope-labeled cells, arginine- and lysine-free McCoy’s medium was supplemented with 200 mg/L l -proline and either l -lysine (Lys0) together with l -arginine (Arg0), l -lysine- 2 H 4 (Lys4) with l -arginine-U- 13 C 6 (Arg6) or l -lysine-U- 13 C 6 - 15 N 2 (Lys8) with l -arginine-U- 13 C 6 - 15 N 4 (Arg10) at final concentrations of 28 mg/L for the arginine and 146 mg/L for the lysine until fully metabolically labeled. The extent of isotope incorporation was assessed using an R-script as described . Cell lysates were prepared, quantified, subjected to SDS PAGE and in-gel tryptic digest as described previously .…”

Section: Materials and Methodsmentioning

confidence: 99%

“…For phosphopeptide (pSer/Thr/Tyr) isolation, we used filter-aided sample preparation (FASP) followed by fractionation using strong cation exchange (SCX) chromatography and TiO 2 -based phosphopeptide isolation (based on refs and and described previously in refs and ). In parallel, quantitative SW48 isogenic cell-line proteome analyses were carried out by resolving a 50 μg aliquot of each SILAC mixture by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on a 4–12% NuPAGE gel (Invitrogen), prior to protein visualization by Colloidal Blue staining (Invitrogen).…”

Section: Materials and Methodsmentioning

confidence: 99%

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Differential Reprogramming of Isogenic Colorectal Cancer Cells by Distinct Activating KRAS Mutations

et al. 2015

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Oncogenic mutations of Ras at codons 12, 13, or 61, that render the protein constitutively active, are found in ∼16% of all cancer cases. Among the three major Ras isoforms, KRAS is the most frequently mutated isoform in cancer. Each Ras isoform and tumor type displays a distinct pattern of codon-specific mutations. In colon cancer, KRAS is typically mutated at codon 12, but a significant fraction of patients have mutations at codon 13. Clinical data suggest different outcomes and responsiveness to treatment between these two groups. To investigate the differential effects upon cell status associated with KRAS mutations we performed a quantitative analysis of the proteome and phosphoproteome of isogenic SW48 colon cancer cell lines in which one allele of the endogenous gene has been edited to harbor specific KRAS mutations (G12V, G12D, or G13D). Each mutation generates a distinct signature, with the most variability seen between G13D and the codon 12 KRAS mutants. One notable example of specific up-regulation in KRAS codon 12 mutant SW48 cells is provided by the short form of the colon cancer stem cell marker doublecortin-like Kinase 1 (DCLK1) that can be reversed by suppression of KRAS.

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“…Incorporation efficiency of SILAC labeling was analyzed using written R script as described in [ 76 ] for all peptides and for peptides with each isotope separately as shown in Supplementary Figure S4 .…”

Section: Methodsmentioning

confidence: 99%

(Phospho)proteomic Profiling of Microsatellite Unstable CRC Cells Reveals Alterations in Nuclear Signaling and Cholesterol Metabolism Caused by Frameshift Mutation of NMD Regulator UPF3A

Michalak

Katzenmaier

Roeckel

et al. 2020

IJMS

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DNA mismatch repair-deficient colorectal cancers (CRCs) accumulate numerous frameshift mutations at repetitive sequences recognized as microsatellite instability (MSI). When coding mononucleotide repeats (cMNRs) are affected, tumors accumulate frameshift mutations and premature termination codons (PTC) potentially leading to truncated proteins. Nonsense-mediated RNA decay (NMD) can degrade PTC-containing transcripts and protect from such faulty proteins. As it also regulates normal transcripts and cellular physiology, we tested whether NMD genes themselves are targets of MSI frameshift mutations. A high frequency of cMNR frameshift mutations in the UPF3A gene was found in MSI CRC cell lines (67.7%), MSI colorectal adenomas (55%) and carcinomas (63%). In normal colonic crypts, UPF3A expression was restricted to single chromogranin A-positive cells. SILAC-based proteomic analysis of KM12 CRC cells revealed UPF3A-dependent down-regulation of several enzymes involved in cholesterol biosynthesis. Furthermore, reconstituted UPF3A expression caused alterations of 85 phosphosites in 52 phosphoproteins. Most of them (38/52, 73%) reside in nuclear phosphoproteins involved in regulation of gene expression and RNA splicing. Since UPF3A mutations can modulate the (phospho)proteomic signature and expression of enzymes involved in cholesterol metabolism in CRC cells, UPF3A may influence other processes than NMD and loss of UPF3A expression might provide a growth advantage to MSI CRC cells.

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Chapter 8. Quantitative LC-MS of Proteins

Cited by 6 publications

References 0 publications

The importance of the ZBED6‐IGF2 axis for metabolic regulation in mouse myoblast cells

The importance of the ZBED6‐IGF2 axis for metabolic regulation in mouse myoblast cells

Differential Reprogramming of Isogenic Colorectal Cancer Cells by Distinct Activating KRAS Mutations

(Phospho)proteomic Profiling of Microsatellite Unstable CRC Cells Reveals Alterations in Nuclear Signaling and Cholesterol Metabolism Caused by Frameshift Mutation of NMD Regulator UPF3A

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