ArticleNature protocols2016
Six alternative proteases for mass spectrometry-based proteomics beyond trypsin.
Article in Nature protocols, 2016. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 201 papers, 1 of them a synthesis that pooled it.
What it found
Each row is one number read from the abstract, on the scale the paper reported it, with its interval. Left of the dashed line favours the treatment, right favours the comparator. Under each row is the sentence it came from. New to these charts? A ten-minute tutorial.
The abstract states no effect estimate the extractor could read, or names no intervention and outcome on the map, so this paper lights no cell and moves no belief. It is still indexed, cited and linked below.
The trial behind it
Trials whose registry record cites this paper, or whose number appears in the abstract. A trial that started after this paper was published is citing it as background, not reporting it.
Neither the registry nor the abstract names a trial number. If this is a trial report, that itself is worth knowing.
Who cites it
201 citing papers in PubMed, 1 synthesis or guideline pooled it.
- Estimating the total number of phosphoproteins and phosphorylation sites in eukaryotic proteomes.GigaScience · 2017Pooled it
- Seryl phosphorylation modulates chaperonin function of Mycobacterial chaperonin, Cpn60.1.Cell stress & chaperones · 2026Article
- SIMPATICO-Streamlined Isolation of the N-Terminome via Phosphonate Tagging and Coordination-Based Depletion.Molecular & cellular proteomics : MCP · 2026Article
- Protein digestion using a cysteine-specific backbone cleavage reagent.RSC chemical biology · 2026Article
- Evolution of an amphibian-specific family of epidermal differentiation-associated genes.Molecular biology and evolution · 2026Article
- Pan-cancer proteogenomic interrogation of the ubiquitin-proteasome system.Cell death and differentiation · 2026Article
- Recent advancements in sample pretreatment methods for glycoproteomics.Biophysics reports · 2026Article
- Article
- Optimized Digestion Conditions for Membrane Protein Footprinting and Mass Spectrometry Analysis.Membranes · 2026Article
- A Flexible Proteomic Approach to A2 Bovine Milk Authentication Through Tryptic, Thermolytic, and Peptic Proteotypic Peptides Elucidated by Routine LC-MS Analysis.Journal of agricultural and food chemistry · 2026Article
- Integrating mass spectrometry with Nanopore direct RNA sequencing forbioRxiv : the preprint server for biology · 2026Article
- IsoPepTracker: An interactive web application for peptide-driven isoform analysis.PLoS computational biology · 2026Article
- RNase 4 improves bottom-up modification mapping ofbioRxiv : the preprint server for biology · 2026Article
- Pan-cancer proteogenomic interrogation of the Ubiquitin Proteasome System.bioRxiv : the preprint server for biology · 2026Article
- Hydrogen/deuterium exchange mass spectrometry analysis of ribosome-nascent chain complexes to study protein biogenesis at the peptide level.Nature protocols · 2026Article
- Deep coverage and extended sequence reads obtained with a single archaeal protease expedite de novo protein sequencing by mass spectrometry.Cell systems · 2026Article
- Harnessing toxin-mediated ribosome stalling as a complementary tool to annotate bacterial ORFs.Nucleic acids research · 2026Article
- Integration of alternative fragmentation techniques into standard LC-MS workflows using a single deep learning model enhances proteome coverage.Nature methods · 2026Article
- Recent Developments and Applications of Capillary and Microchip Electrophoresis in Proteomics and Peptidomics (2023-2025).Journal of separation science · 2026Review
- Comparative Analysis of Lysine-Specific Peptidases for Optimizing Proteomics Workflows.Journal of proteome research · 2026Article
141 more citing papers are in PubMed but not listed here.
Corrections and comments
PubMed lists nothing against this paper. Absence here is not a guarantee, only a check that was made.
Authors and funding
4 authors.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Protein digestion using a dedicated protease represents a key element in a typical mass spectrometry (MS)-based shotgun proteomics experiment. Up to now, digestion has been predominantly performed with trypsin, mainly because of its high specificity, widespread availability and ease of use. Lately, it has become apparent that the sole use of trypsin in bottom-up proteomics may impose certain limits in our ability to grasp the full proteome, missing out particular sites of post-translational modifications, protein segments or even subsets of proteins. To overcome this problem, the proteomics community has begun to explore alternative proteases to complement trypsin. However, protocols, as well as expected results generated from these alternative proteases, have not been systematically documented. Therefore, here we provide an optimized protocol for six alternative proteases that have already shown promise in their applicability in proteomics, namely chymotrypsin, LysC, LysN, AspN, GluC and ArgC. This protocol is formulated to promote ease of use and robustness, which enable parallel digestion with each of the six tested proteases. We present data on protease availability and usage including recommendations for reagent preparation. We additionally describe the appropriate MS data analysis methods and the anticipated results in the case of the analysis of a single protein (BSA) and a more complex cellular lysate (Escherichia coli). The digestion protocol presented here is convenient and robust and can be completed in ∼2 d.
Indexed as
Identifiers
27123950What OpenQuestion holds
Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.