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. 2023 May 8;14:2642. doi: 10.1038/s41467-023-38171-8

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — a Scheme of cell-selective proteomics workflows: The methionyl-tRNA synthetase^L274G (MetRS*) activates azidonorleucine (Anl) by loading it onto methionyl-tRNAs. MetRS*-expressing cells incorporate Anl as a methionine substitute into newly synthesized proteins. Lentivirally transduced primary MetRS*-expressing or wild-type (Ctrl) PDAC cells isolated from mouse tumors with a conditional pancreatic expression of Kras^G12D were grown for 8 h in Met-depleted medium supplemented with 4 mM Anl. 1 × 10⁷ MetRS* and Ctrl cells were processed by DST enrichment, DBCO enrichment, and our improved alkyne-agarose CuAAC enrichment protocols (n = 3, workflow replicates). b Peptide yields (mean ± SD) determined by absorbance at 280 nm after enrichment, digestion, and solid phase peptide extraction. c Identified protein groups (mean ± SD) after MS-based analysis using 2 h chromatographic gradient length and data-dependent acquisition (DDA). d Intensity ratios of proteins identified in MetRS* and Ctrl samples. Counts of overlapping identifications with ratios are indicated. e Specifically enriched protein groups (exclusive or >3-fold higher intensity compared to Ctrl samples) identified after alkyne-agarose enrichment and single run DDA, DDA analysis of 16 fractions separated by offline high-pH reverse phase chromatography, or single run data-independent acquisition (DIA) (mean ± SD, fractionation n = 1, single shots n = 3, workflow replicates). The latter was used for all further experiments. f Scheme of cell-selective secretomics workflow: MetRS* and Ctrl 8661 PDAC cells were cultured for 8 h in 5% FBS containing Met-depleted medium with 4 mM Anl (n = 3, workflow replicates). MetRS*-expressing cell-derived Anl-proteins were enriched from cell supernatants after buffer exchange and concentration. g Specifically-enriched PDAC cell-released proteins ranked by label-free quantification (LFQ) intensity. Proteins with cytokine function are indicated. Source data are provided as a Source Data file.