Precise Characterization of KRAS4B Proteoforms by Combining Immunoprecipitation with Top-Down Mass Spectrometry

Adams, Lauren; DeHart, Caroline J.; Kelleher, Neil L.

doi:10.1007/978-1-0716-1190-6_3

Cited by 6 publications

(6 citation statements)

References 13 publications

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“…Briefly, cells or homogenized colorectal tumor tissue were lysed in a buffer containing two nonionic detergents. RAS was then immunoenriched from the lysates, with eluted proteoforms desalted by solid-phase extraction prior to LC coupled with a Q-Exactive HF mass spectrometer ( 42 ). After a survey for an initial stage of proteoform discovery for each sample type, we generated a list of proteoforms for targeted analysis and extensive characterization.…”

Section: Resultsmentioning

confidence: 99%

“…Proteoforms are first detected in discovery mode (intact MS1) and then further characterized in targeted mode using tandem mass spectrometry (tMS1/tMS2). Key differences in the protocol compared with those previously published ( 41 , 42 ) are denoted by asterisks . IP, immunoprecipitation; TDMS, top–down mass spectrometry.…”

Section: Resultsmentioning

confidence: 99%

“…IP with anti-v-HRAS agarose IP beads (OP01A; MilliporeSigma, Research Resource Identifier [RRID]: AB_437743 ) was performed as previously reported ( 42 ), with the exception of the lysis buffer composition, which now contained 50 mM Tris (pH 7.5), 150 mM NaCl, 1% NP-40 (MilliporeSigma), 1% Triton X-100 (Thermo Fisher Scientific), and 1× final concentration of HALT Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific). IP reactions were performed in triplicate for cell lines.…”

Section: Methodsmentioning

confidence: 99%

“…Our laboratory previously reported a study employing immunoprecipitation coupled with top-down mass spectrometry (IP–TDMS) to characterize 11 KRAS4B proteoforms in isogenic colorectal cancer cell lines and 6 primary colorectal tumors ( 41 ). Here, through an improved IP–TDMS protocol with enhanced limits of detection, we characterized both KRAS4A and KRAS4B proteoforms, including those present at <5% relative abundance ( 41 , 42 ). We deployed this optimized KRAS proteoform assay to a panel of 14 cell lines and 34 colorectal tumor samples.…”

mentioning

confidence: 99%

See 3 more Smart Citations

Mapping the KRAS proteoform landscape in colorectal cancer identifies truncated KRAS4B that decreases MAPK signaling

Adams

DeHart

Drown

et al. 2023

Journal of Biological Chemistry

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Mapping the KRAS proteoform landscape in colorectal cancer identifies truncated KRAS4B that decreases MAPK signaling

Adams

DeHart

Drown

et al. 2023

Journal of Biological Chemistry

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“…In clinical samples, measuring the free target enables a single assay that evaluates target engagement and also compares the target expression levels within the studied population. Several targeted proteomics assays have been developed to quantify RASG12C protein expression in cell line models and frozen tissue samples at baseline and/or after administration of RASG12C inhibitors [ 9 – 16 ].…”

Section: Introductionmentioning

confidence: 99%

Mass spectrometry quantifies target engagement for a KRASG12C inhibitor in FFPE tumor tissue

Chambers,

Chain,

Sweet

et al. 2023

Clin Proteom

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Background Quantification of drug-target binding is critical for confirming that drugs reach their intended protein targets, understanding the mechanism of action, and interpreting dose-response relationships. For covalent inhibitors, target engagement can be inferred by free target levels before and after treatment. Targeted mass spectrometry assays offer precise protein quantification in complex biological samples and have been routinely applied in pre-clinical studies to quantify target engagement in frozen tumor tissues for oncology drug development. However, frozen tissues are often not available from clinical trials so it is critical that assays are applicable to formalin-fixed, paraffin-embedded (FFPE) tissues in order to extend mass spectrometry-based target engagement studies into clinical settings. Methods Wild-type RAS and RASG12C was quantified in FFPE tissues by a highly optimized targeted mass spectrometry assay that couples high-field asymmetric waveform ion mobility spectrometry (FAIMS) and parallel reaction monitoring (PRM) with internal standards. In a subset of samples, technical reproducibility was evaluated by analyzing consecutive tissue sections from the same tumor block and biological variation was accessed among adjacent tumor regions in the same tissue section. Results Wild-type RAS protein was measured in 32 clinical non-small cell lung cancer tumors (622–2525 amol/µg) as measured by FAIMS-PRM mass spectrometry. Tumors with a known KRASG12C mutation (n = 17) expressed a wide range of RASG12C mutant protein (127–2012 amol/µg). The variation in wild-type RAS and RASG12C measurements ranged 0–18% CV across consecutive tissue sections and 5–20% CV among adjacent tissue regions. Quantitative target engagement was then demonstrated in FFPE tissues from 2 xenograft models (MIA PaCa-2 and NCI-H2122) treated with a RASG12C inhibitor (AZD4625). Conclusions This work illustrates the potential to expand mass spectrometry-based proteomics in preclinical and clinical oncology drug development through analysis of FFPE tumor biopsies.

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FLAG-KRAS4B as a Model System for KRAS4B Proteoform and PTM Evaluation by Mass Spectrometry

D’Ippolito,

Scheidemantle,

Smith

et al. 2024

Methods in Molecular Biology

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Precise Characterization of KRAS4B Proteoforms by Combining Immunoprecipitation with Top-Down Mass Spectrometry

Cited by 6 publications

References 13 publications

Mapping the KRAS proteoform landscape in colorectal cancer identifies truncated KRAS4B that decreases MAPK signaling

Mapping the KRAS proteoform landscape in colorectal cancer identifies truncated KRAS4B that decreases MAPK signaling

Mass spectrometry quantifies target engagement for a KRASG12C inhibitor in FFPE tumor tissue

FLAG-KRAS4B as a Model System for KRAS4B Proteoform and PTM Evaluation by Mass Spectrometry

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