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Sequential Digestion with Different Proteases

ABSTRACT: We examined three combinations of different proteases for digestion of proteins from TurboID-STING-expressing cells: trypsin alone, Lys-C followed by trypsin, and Glu-C followed by trypsin. First, we tested whether PTS buffer used to solubilize precipitated proteins is compatible with digestion with these proteases. Next, biotinylated peptides were enriched after digestion with the three combinations of different proteases. We assessed reproducibility of each combination using three technical replicates.

ORGANISM(S): Mus Musculus (mouse)

SUBMITTER: Hidetaka Kosako

PROVIDER: PXD035242 | JPOST Repository | Thu Aug 25 00:00:00 BST 2022

REPOSITORIES: jPOST

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Optimized Workflow for Enrichment and Identification of Biotinylated Peptides Using Tamavidin 2-REV for BioID and Cell Surface Proteomics.

Nishino Kohei K Yoshikawa Harunori H Motani Kou K Kosako Hidetaka H

Journal of proteome research 20220817 9

Chemical or enzymatic biotinylation of proteins is widely used in various studies, and proximity-dependent biotinylation coupled to mass spectrometry is a powerful approach for analyzing protein-protein interactions in living cells. We recently developed a simple method to enrich biotinylated peptides using Tamavidin 2-REV, an engineered avidin-like protein with reversible biotin-binding capability. However, the level of biotinylated proteins in cells is low; therefore, large amounts of cellular ...[more]

PMID: 35979633

Similar Datasets

Sequential Digestion with Different Proteases

Project description:We examined three combinations of different proteases for digestion of proteins from TurboID-STING-expressing cells: trypsin alone, Lys-C followed by trypsin, and Glu-C followed by trypsin. First, we tested whether PTS buffer used to solubilize precipitated proteins is compatible with digestion with these proteases. Next, biotinylated peptides were enriched after digestion with the three combinations of different proteases.

2023-03-04 | PXD032061 | JPOST Repository

Consecutive digestion of archaeological skeletal proteomes with multiple proteases

Project description:Ancient skeletal proteomes are increasingly utilised for phylogenetic and evolutionary analysis. These proteomes are, however, often small and with low sequence coverage. We expand on previous observations which have shown that parallel digestion of Pleistocene skeletal proteomes increases proteome size and protein sequence coverage. Furthermore, we demonstrate that the consecutive digestion of a skeletal proteome using two proteases, particularly Glu-C or chymotrypsin followed by trypsin digestion, enables the recovery of alternative proteome components not reachable through trypsin digestion alone. The sequential utilisation of several proteases is a promising avenue for the study of highly degraded ancient proteomes for phylogenetic purposes.

2024-03-06 | PXD047069 | Pride

Expression data from mammary epithelial cells harvested from 8-9 week old C3H female mice - various isolation conditions

2014-09-05 | GSE61167 | GEO

Expression data from mammary epithelial cells harvested from 8-9 week old C3H female mice - various isolation conditions

Project description:Numerous lineage analysis studies have used FACS sorting as a separation technique prior to measuring mRNA expression patterns. To assess if FACS sorting causes non-specific changes in gene expression, we collected a heterogeneous population of mammary epithelial cells from three biological replicates. We then performed gene expression analysis on each replicate either prior to trypsin digestion, immediately following trypsin digestion, or following both the trypsin digestion and a mock FACS sort. In this dataset, we include gene expression data obtained from isolated murine mammary epithelial cells either prior to trypsin digestion, immediately following trypsin digestion, or following the trypsin digestion and a mock FACS sort. Nine total samples were analyzed (3 biological replicates of three experimental conditions). Using LIMMA packages gene-wise comparisons were made between untrypsinized and trypsinized replicates as well as between trypsinized and mock FACS sorted replicates using and Pâ¤0.05 and a â¥1.5-fold difference between conditions.

2014-09-05 | E-GEOD-61167 | biostudies-arrayexpress

Promiscuous gene expression in human medullary thymic epithelial cell subsets

Project description:Different human mTEC subsets (MUC1, CEACAM5 and SGLT1) were purified by sequential enzymatic digestion (collagenase/dispase, trypsin) followed by enrichment using magnetic beads (CD45 beads, Miltenyi Biotech) and FACS sorting. Cells of the surface phenotype CD45-, CDR2-, EpCAM+ were further subdivided into MUC1+/MUC1-, CEACAM5+/CEACAM5- and SGLT1+/SGLT1- fractions. RNA was isolated using μMACS™ SuperAmp™ protocol (Miltenyi Biotec) and hybridized to Illumina Whole-Genome Expression Beadchips. Gene expression of Antigen-positive and Antigen-negative mTEC subsets was compared.

2013-08-31 | GSE49625 | GEO

Proteomics Using Proteases Alternative to Trypsin Benefits from Sequential Digestion with Trypsin

Project description:High specificity and ease of use make trypsin the most used enzyme in proteomics. Proteases with complementary cleavage specificity to trypsin have been applied to obtain additional data. However, use of proteases with broad specificity proved especially challenging. In this work, we analyzed the characteristics of five protease alternatives to trypsin for protein identification and sequence coverage when applied to S. pombe whole cell lysates. The specificity of the protease heavily impacted on the number of proteins identified. Proteases with higher specificity let to the identification of more proteins than proteases with lower specificity. However, AspN, GluC, chymotrypsin and proteinase K largely benefited from being paired with trypsin in sequential digestion, as had been shown by us for elastase before. In the most extreme case, the addition of trypsin to a proteinase K digest increased the number of identified proteins by 524 %. Also, AspN (82 %) and GluC (74 %) protein identifications largely improved following the additional digestion with trypsin. In general, protein identifications improved most over the use of the single protease when the enzymes followed on an initial digestion with trypsin. In the most extreme case, the sequential digest with trypsin and AspN yielded even higher number of protein identifications than digesting with trypsin alone.

2021-06-07 | PXD017321 | Pride

Quantitative assessment of the effects of trypsin digestion methods on immunoprecipitation-mass spectrometry-based protein-protein interaction analysis

Project description:Immunoprecipitation-mass spectrometry (IP-MS) has become the method of choice for discovering protein-protein interaction (PPI) under native conditions. The success of the IP-MS depends on the efficiency of trypsin digestion and recovery of the tryptic peptides for MS analysis. Several different protocols have been used for trypsin digestion of protein complexes in IP-MS studies, but no systematic studies have been conducted on the impact of trypsin digestion conditions on the identification of PPI. Here, we identified the interactome of transcription factor p65 (RelA) to test five distinct trypsin digestion methods (two using “on-bead”, two using “in-solution”, and one using “in-gel” protocols) and determined which method yielded the best interactome coverage. Our study indicates that high-abundance RelA interactors can be universally identified by all five methods, but the identification of low-abundance RelA interactors is significantly affected by the choice of trypsin digestion methods. We found that eluting RelA complex with sodium dodecyl sulfate (SDS) and followed by filter-aided sample preparation (FASP)/in-solution digestion is the best among the methods that were tested. We also found that different digestion protocols influences the selected reaction monitoring (SRM)-MS quantification of PPIs, suggesting that optimization of trypsin digestion condition may be required for robust study of targeted analysis of PPIs.

2017-07-25 | PXD005984 | Pride

Quantitative assessment of the effects of trypsin digestion methods on immunoprecipitation-mass spectrometry-based protein-protein interaction analysis - part2

2017-07-26 | PXD006739 | Pride

ABIN1 is an signal-induced autophagy adaptor counteracting LUBAC-mediated linear ubiquitination

Project description:ABIN1 KO RAW264.7 cells and stably reconstituted with 3×FLAG-ABIN1 WT or D485N were subjected to mass spectrometric analysis. Three biological replicates were prepared for each condition. Cells were stimulated for 3 hours with 100 ng/ml LPS. ABIN1 was immunoprecipitated with 30 uL anti-FLAG M2 Magnetic Beads (M8823, Sigma-Aldrich) for 2 h at 4℃. Immunoprecipitated sample was eluted by incubation at 90°C for 5 min in 100 uL PTS buffer. Each sample was split into 3 tubes for protein digestion with Lys-C and trypsin, chymotrypsin, or Glu-C.

2022-02-17 | PXD024864 | JPOST Repository

RNAseq of trachea, bronchi and lungs of mice to investigate expression and distribution of trypsin-like proteases in the murine lower airways.

Project description:RNAseq of trachea, bronchi and lungs of mice to investigate expression and distribution of trypsin-like proteases in the murine lower airways in order to identfy potential host cell proteases capable of cleaving influenza surface glycoprotein hemaglutinine (HA) during infection.

2020-03-27 | E-MTAB-8573 | biostudies-arrayexpress

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