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Protein Digestion

Protein Digestion is the biological process of breaking down complex protein molecules into smaller peptides and amino acids.
This crucial process occurs in the gastrointestinal tract, facilitating the absorption and utilization of essential nutrients.
Proteolytic enzymes, such as pepsin, trypsin, and chymotrypsin, play a key role in this process, cleaving peptide bonds and liberating individual amino acids.
Proper protein digestion is vital for maintaining overall health, supporting muscle growth, and ensuring efficient nutrient uptake.
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Most cited protocols related to «Protein Digestion»

1
Quantitative Proteomics of Yeast Strains
Cited 73 times
We chose Saccharomyces cerevisae strains from the haploid
MATalpha collection (BY4742 MATα his3Δ1 leu2Δ0
lys2Δ0 ura3Δ0). Cultures were grown in standard
yeast-peptone-dextrose (YPD) media to an optical density (OD) of 0.6/mL and then
harvested. Cell lysis, protein digestion, and tandem mass tag (TMT) labeling of the
yeast cultures were performed as described previously [12 (link), 14 ].
Very briefly, cells were lysed via bead beating in 8 M urea. Proteins were
chloroform-methanol precipitated and digested with LysC overnight and trypsin for 6
hrs. Peptides were labeled with TMT10-plex reagents, such that peptides from the
Δmet6 strain replicates were conjugated to tags 126,
127N, 127C, the Δpfk2 strain replicates with tags 128N,
128C, 129N, and the Δura2 strain replicates with tags 129C,
130N, 130C. The sample was mixed, desalted via SepPak, and dried via vacuum
centrifugation. The sample was reconstituted in 5% acetonitrile and
5% formic acid for LC-MS/MS processing. For each analysis, 0.1-1 μg
of the TKO standard was loaded onto the C18 capillary column using a Proxeon
NanoLC-1000 UHPLC. Mass spectrometric data were collected on an Orbitrap Fusion or
Lumos mass spectrometer, as described previously [15 ], but with no off-line fractionation and
analyzed with only 45 min liquid chromatography gradients. Mass spectra were
processed with a SEQUEST-based in-house software pipeline [16 (link)]. PSMs were identified, quantified, and
collapsed to a 1% peptide false discovery rate (FDR) and then collapsed
further to a final protein-level FDR of 1% [17 (link), 18 (link)].
Proteins and peptides lists with associated TMT signal-to-noise values were exported
from our in-house Sequest-based software suite for further analysis in Microsoft
Excel, GraphPad Prism, and BoxPlotR [19 (link)]. Please refer to Supplementary Material for expanded
experimental methods.
Paulo J.A., O'Connell J.D, & Gygi S.P. (2016). A Triple Knockout (TKO) Proteomics Standard for Diagnosing Ion Interference in Isobaric Labeling Experiments. Journal of the American Society for Mass Spectrometry, 27(10), 1620-1625.
Publication 2016
acetonitrile Capillaries Cells formic acid Fractionation, Chemical Glucose glutamate carboxypeptidase II, human Liquid Chromatography Mass Spectrometry Methanol Peptides Peptones prisma Protein Digestion Proteins Saccharomyces cerevisiae Staphylococcal Protein A Strains Tandem Mass Spectrometry Trypsin Urea Vision
2
CUT&RUN Profiling of Transcription Factors and Histone Modifications
Cited 41 times
CUT&RUN was performed as previously described [10 (link)]. Briefly, cells were washed with Wash Buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine and one Roche Complete protein inhibitor tablet per 50 mL), bound to Concanavalin A-coated magnetic beads and incubated with primary antibody diluted in wash buffer containing 0.05% digitonin (Dig Wash) overnight at 4 °C. Cells were then washed and incubated with protein A-MNase (pA-MN) for 1 h at 4 °C. Slurry was washed again and placed on an ice-cold block and incubated with Dig Wash containing 2 mM CaCl2 to activate pA-MN digestion. After digestion for 30 min, one volume of 2× stop buffer (340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 0.05% Digitonin, 0.05 mg/mL glycogen, 5 µg/mL RNase A, 2 pg/mL heterologous spike-in DNA) was added to stop the reaction, and fragments were released by 30-min incubation at 37 °C. Samples were centrifuged 5 min at 16,000×g, and supernatant was recovered and DNA extracted via phenol–chloroform extraction and ethanol precipitation. Resulting DNA was used as input for library preparation as previously described [10 (link)]. Antibodies used for CUT&RUN in this study were as follows: rabbit anti-Sox2 (Abcam ab92494); rabbit anti-FoxA2 (Millipore 07-633); Guinea-Pig anti-rabbit IgG (antibodies online ABIN101961); rabbit anti-H3K4me2 (Millipore 07-030); rabbit anti-H3K4me3 (Active Motif 39159); rabbit anti-H3K27me3 (Cell Signaling Technologies CST9733); and rabbit anti-CTCF (Millipore 07-729).
Meers M.P., Tenenbaum D, & Henikoff S. (2019). Peak calling by Sparse Enrichment Analysis for CUT&RUN chromatin profiling. Epigenetics & Chromatin, 12, 42.
Publication 2019
anti-IgG Antibodies Buffers Cavia porcellus Cells Chloroform Cold Temperature Concanavalin A CTGF protein, human Digestion Digitonin DNA Library Edetic Acid Egtazic Acid Ethanol Glycogen HEPES histone H3 trimethyl Lys4 Immunoglobulins Phenol Protein Digestion Proteins Rabbits Ribonucleases Sodium Chloride SOX2 protein, human Spermidine Staphylococcal Protein A Tablet
3
Biotinylated Protein Enrichment and SILAC Quantification
Cited 16 times

Protocol full text hidden due to copyright restrictions

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Publication 2014
Biotin Buffers DNA Replication Isotopes Liquid Chromatography Mass Spectrometry Protein Digestion Proteins Radioimmunoprecipitation Assay Staphylococcal Protein A Streptavidin Tromethamine Urea
4
Intracellular Metabolite Analysis via GC-MS
Cited 12 times
For GC-MS analysis of intracellular metabolites, cells were grown to about 1
million cells/0.5 mg cell protein per culture well. Medium was removed and saved
for analysis, cells were washed quickly 3 times with cold PBS, and 0.45 ml cold
methanol (50% v/v in water with 20 μM L-norvaline as internal standard)
was added to each well. Culture plates were transferred to dry ice for 30 min.
After thawing on ice, the methanol extract was transferred to a microcentrifuge
tube (the described treatment disrupted cells without the necessity of
scraping). Chloroform (0.225 ml) was added, the tube was vortexed and
centrifuged at 10,000g for 5 min at 4°C. The upper layer was dried in a
centrifugal evaporator and derivatized with 30 μl O-isobutylhydroxylamine
hydrochloride (20 mg/ml in pyridine, TCI) for 20 min at 80°C, followed by
30 μl
N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide
(Sigma or Regis) for 60 min at 80°C. After cooling, the derivatization
mixture was transferred to an autosampler vial for analysis.
For digestion of cellular proteins, cells washed as above (while still attached
to plates) were lysed in 0.6 ml 10 mM tris-HCl, pH 7.3 containing 1 mM EDTA, 1%
Triton X100 and 0.4 mM L-norvaline. A small fraction (20 μl) was dried
and digested for 18 h with 200 μl 6N HCl. After drying under nitrogen,
the digest was derivatized for GC-MS as above.
For GC-MS analysis of medium, 40 μl of medium was mixed with 0.4 ml cold
methanol (50% v/v in water with 20 μM L-norvaline as internal standard).
The methanolic extract was counter-extracted with 0.2 ml chloroform, dried, and
derivatized as for cell extracts.
GC-MS protocols were similar to those described before [3 (link)], except a modified temperature gradient was used for GC:
Initial temperature was 130°C, held for 4 min, rising at 6°C/min
to 243°C, rising at 60°C/min to 280°C, held for 2 min. Data
were corrected for natural 13C labeling as before [3 (link)]. Metabolites were quantified against
varied amounts of standard mixtures run in parallel and data were analyzed using
Metaquant [35 (link)]. Quantities were corrected
for recovery using the L-norvaline internal standard. Glutamine uptake from
medium and lactate secretion into medium were measured using a YSI model 7100
enzyme analyzer rather than by GC-MS.
Ratnikov B., Aza-Blanc P., Ronai Z.A., Smith J.W., Osterman A.L, & Scott D.A. (2015). Glutamate and asparagine cataplerosis underlie glutamine addiction in melanoma. Oncotarget, 6(10), 7379-7389.
Publication 2015
ARID1A protein, human Cell Culture Techniques Cell Extracts Cells Chloroform Cold Temperature Dry Ice Edetic Acid Gas Chromatography-Mass Spectrometry Glutamine Lactate Methanol Nitrogen norvaline Protein Digestion Proteins Protoplasm pyridine secretion TERT protein, human Triton X-100 Tromethamine
5
Quantitative Proteomic Workflow Analysis
Cited 12 times
The
raw files acquired by the MS system were processed using the Proteome
Discoverer platform (version 1.4, Thermo Scientific). An integrated
workflow using the algorithms Sequest HT and Mascot (version 2.4,
Matrix Science) was employed. Either a human UniProtKB database (Release
2013_6; 88 295 human sequences) or a database consisting of
the aforementioned human proteins and all protein sequences derived
from 21 microbial genomes (Supporting Information Table S-1) were used. The latter database was used to identify human
and microbial proteins present in UP samples. MS search parameters
similar to published previously27 are described
in detail in Supporting Information. For
protein quantification of the data sets, the MaxQuant software suite
(version 1.4.2) was used.32 (link) Most of the
default settings provided in this software suite were accepted, and
data were processed using both the label-free quantitation (LFQ) and
the intensity-based absolute quantitation (iBAQ) tools. The LFQ algorithms
provide relative quantification of the integrated MS1 peak
areas from the high resolution MS data. The iBAQ algorithms sum the
integrated peak intensities of the peptide ions for a given protein
divided by the number of theoretically observable peptides, which
are calculated by in silico digestion of protein sequences including
all fully tryptic peptides with a length of 6–30 amino acids.33 (link)
Yu Y., Suh M.J., Sikorski P., Kwon K., Nelson K.E, & Pieper R. (2014). Urine Sample Preparation in 96-Well Filter Plates for Quantitative Clinical Proteomics. Analytical Chemistry, 86(11), 5470-5477.
Publication 2014
Amino Acids Amino Acid Sequence Genome Homo sapiens Ions NR4A2 protein, human Peptides Protein Digestion Proteins Trypsin

Most recents protocols related to «Protein Digestion»

1
MS-Based Peptide Profiling Protocol
The peptides were analyzed using a MS shotgun proteomics technique. This technique allows a sensitive bottom-up approach that consists of separating peptides resulting from protein digestion by liquid HPLC followed by tandem mass spectrometry (MS/MS). Samples were run on a Bruker timsTOF Pro mass spectrometer connected to an Evosep One liquid chromatography system. Tryptic peptides were resuspended in 0.1% formic acid, and each sample was loaded onto an Evosep tip. The Evosep tips were placed in position on the Evosep One, in a 96-tip box. The autosampler is configured to pick up each tip, elute, and separate the peptides using a set chromatography method (Bache et al, 2018 (link)). The chromatography buffers used were buffer B (99.9% acetonitrile, 0.1% formic acid) and buffer A (99.9% water, 0.1% formic acid). All solvents are LC-MS–grade.
The mass spectrometer was operated in a positive ion mode with a capillary voltage of 1,500 V, dry gas flow of 3 liters/min, and a dry temperature of 180°C. All data were acquired with the instrument operating in a trapped ion mobility spectrometry mode. Trapped ions were selected for ms/ms using parallel accumulation–serial fragmentation. A scan range of (100–1,700 m/z) was performed at a rate of 5 parallel accumulation–serial fragmentation MS/MS frames to 1 MS scan with a cycle time of 1.03 s (Meier et al, 2018 (link)).
The data analysis was done using MaxQuant software (Cox & Mann, 2008 (link)). The raw data were searched against the Homo sapiens subset of the UniProt/SwissProt database (reviewed) with the search engine MaxQuant (release 2.0.3.0). Specific parameters for trapped ion mobility spectrometry data–dependent acquisition were used: Fixed Mod: carbamidomethylation; Variable Mods: methionine, oxidation; Trypsin/P digest enzyme: maximum two missed cleavages; Precursor mass tolerances: 10 ppm; Peptide FDR: 1%; and Protein FDR: 1%. The normalized protein intensity of each identified protein was used for label-free quantitation (LFQ) using the MaxLFQ algorithm (Cox et al, 2014 (link)).
The MS proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (Perez-Riverol et al, 2022 (link)) partner repository with the dataset identifier PXD035399.
Ternet C., Junk P., Sevrin T., Catozzi S., Wåhlén E., Heldin J., Oliviero G., Wynne K, & Kiel C. (2023). Analysis of context-specific KRAS–effector (sub)complexes in Caco-2 cells. Life Science Alliance, 6(5), e202201670.
Publication 2023
acetonitrile Buffers Capillaries Chromatography Cytokinesis Enzymes formic acid High-Performance Liquid Chromatographies Homo sapiens Immune Tolerance Ion Mobility Spectrometry Ions Liquid Chromatography Mass Spectrometry Methionine Multiple Organ Failure Peptides Protein Digestion Proteins Radionuclide Imaging Reading Frames Solvents Tandem Mass Spectrometry Trypsin
2
SILAC-Based Protein Quantification and Digestion
The protein concentration of the SILAC cell lysates was determined using the bicin chonicic acid assay (Pierce). Digestion of the proteins was performed using the Filter-Aided Sample Preparation (FASP) method [108 (link)], for which equal amounts (900 μg) of mock- and virus-infected cell lysates were mixed and DTT was added to a final concentration of 50 mM, followed by a 5-min incubation at 70°C. Samples were loaded on two 15-ml 30 kDa Microcon filter devices (Millipore), which were washed twice with 8 M urea 0.1 M Tris pH 8.5, while cysteines were alkylated with 50 mM iodoacetamide in the same buffer. Samples were washed 3 times with 8 M urea, 0.1 M Tris pH 8. Proteins were digested overnight at room temperature using 20 ug endoLysC (Wako Pure Chemical Industries) in the same buffer per filter device. The sample was diluted fourfold with 50 mM ammonium bicarbonate pH 8.4 containing 20 ug trypsin (Worthington Chemical Corporation), and digested for 4 h at room temperature. Peptides were collected by centrifugation, acidified to a final percentage of 1% TFA, and desalted using solid phase extraction. Peptides were eluted in 20/80/0.1 (v/v/v) of milliQ/acetonitrile (ACN) (Actu-All Chemicals)/trifluoric acid (TFA) (Sigma-Aldrich).
Treffers E.E., Tas A., Scholte F.E., de Ru A.H., Snijder E.J., van Veelen P.A, & van Hemert M.J. (2023). The alphavirus nonstructural protein 2 NTPase induces a host translational shut-off through phosphorylation of eEF2 via cAMP-PKA-eEF2K signaling. PLOS Pathogens, 19(2), e1011179.
Publication 2023
acetonitrile Acids ammonium bicarbonate Biological Assay Buffers Cells Centrifugation Cysteine Iodoacetamide Medical Devices Peptides Protein Digestion Proteins Solid Phase Extraction Tromethamine Trypsin Urea Virus
3
Dual-Enzyme Protein Digestion Protocol
Following enrichment, 60 µl 4 M urea/50 mM Tris-HCl was added to each well. Afterwards, reduction of proteins was executed for 1h in the thermomixer (800 rpm, 37°C) in the presence of 8 mM DTT, followed by alkylation for 30 min in the dark (37°C) in the presence of 22 mM IAM. Digestion was started by addition of Lys-C at 0,4 µg/well and left overnight (thermomixer 37°C, 800 rpm, dark). The next day, 50 mM Tris-HCl was added until urea concentration dropped below 1 M. Chymotrypsin was added at 1 µg/well and digestion of the proteins continued in the thermomixer (4 h, 37°C, 800 rpm). Finally, digestion was stopped in the presence of 1% FA.
Frans G., Dillaerts D., Dehaemers T., Van Elslande J., De Leeuw J., Boon L., Maes W., Callewaert N., Calcoen B., Ancheva L., Cockx M., Geukens N., Arat K., Derua R., Vermeersch P., Carpentier S.C, & Bossuyt X. (2023). Complementarity determining regions in SARS-CoV-2 hybrid immunity. Frontiers in Immunology, 14, 1050037.
Publication 2023
Alkylation Chymotrypsin Digestion Protein Digestion Proteins Tromethamine Urea
4
Nickel Affinity Purification and Tryptic Digestion
For
each biological replicate, 9 mg of total cell extract (TCE) were incubated
with 360 μL of Ni-NTA beads (Qiagen) at 4 °C for 16 h.
The beads were subsequently washed once with 12 mL of Ni-NTA denaturing
incubation buffer, five times with 12 mL of Ni-NTA denaturing washing
buffer (8 M urea, 100 mM NaH2PO4, 20 mM imidazole,
5 mM 2-mercaptoethanol, 20 mM chloroacetamide, 10 mM Tris–HCl
pH = 6.3), and twice with 10 mL of 50 mM ammonium bicarbonate. Protein
quantification on beads was conducted using micro Bradford assay (Bio-Rad).
Protein digestion on beads was carried out using a ratio 1:20 of sequencing-grade
modified trypsin (Promega):protein extract in 50 mM ammonium bicarbonate
at 37 °C overnight. To quench the reaction, 1% trifluoroacetic
acid (TFA) was added. Sample desalting was performed on hydrophilic–lipophilic
balance (HLB) cartridges (3 cc, 60 mg) (Waters) and eluted in low
protein binding collection tubes (Thermo Fisher Scientific). Finally,
samples were dried down by a Speed Vac prior to LC–MS/MS analysis.
Li C., Boutet A., Pascariu C.M., Nelson T., Courcelles M., Wu Z., Comtois-Marotte S., Emery G, & Thibault P. (2023). SUMO Proteomics Analyses Identify Protein Inhibitor of Activated STAT-Mediated Regulatory Networks Involved in Cell Cycle and Cell Proliferation. Journal of Proteome Research, 22(3), 812-825.
Publication 2023
2-Mercaptoethanol Ammonium ammonium bicarbonate Biological Assay Biopharmaceuticals Buffers Cell Extracts chloroacetamide DNA Replication imidazole Promega Protein C Protein Digestion Tandem Mass Spectrometry Tromethamine Trypsin Urea
5
Phosphoproteome Trypsin Digestion
The trypsin digestion of the phosphoproteome sequence is discussed in the Protein digestion section.
Zhou M., Li Y., Yan Y., Gao L., He C., Wang J., Yuan Q., Miao L., Li S., Di Q., Yu X, & Sun M. (2023). Proteome and phosphoproteome analysis of 2,4-epibrassinolide-mediated cold stress response in cucumber seedlings. Frontiers in Plant Science, 14, 1104036.
Publication 2023
Digestion Protein Digestion Trypsin

Top products related to «Protein Digestion»

1
Trypsin by Promega
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Trypsin is a serine protease enzyme that is commonly used in cell culture and molecular biology applications. It functions by cleaving peptide bonds at the carboxyl side of arginine and lysine residues, which facilitates the dissociation of adherent cells from cell culture surfaces and the digestion of proteins.
2
Sequencing grade trypsin by Promega
Sourced in United States, Germany, United Kingdom, Italy, France, Spain, Switzerland
Sequencing grade trypsin is a proteolytic enzyme used to cleave peptide bonds in protein samples, primarily for use in protein sequencing applications. It is purified to ensure high-quality, consistent performance for analytical processes.
3
Sequencing grade modified trypsin by Promega
Sourced in United States, United Kingdom, Germany, France, Switzerland, Japan, China, Sweden
Sequencing grade modified trypsin is a protease enzyme used for the digestion of proteins prior to mass spectrometry analysis. It is designed to provide consistent, high-quality peptide digestion for protein identification and characterization.
4
Dithiothreitol (dtt) by Merck Group
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Dithiothreitol (DTT) is a reducing agent commonly used in biochemical and molecular biology applications. It is a small, water-soluble compound that helps maintain reducing conditions and prevent oxidation of sulfhydryl groups in proteins and other biomolecules.
5
High intensity ultrasonic processor by Ningbo Scientz Biotechnology
Sourced in China, Germany
The High intensity ultrasonic processor is a laboratory equipment designed to generate high-frequency mechanical vibrations. It utilizes piezoelectric transducers to convert electrical energy into ultrasonic energy, which can be used for various applications in scientific research and industrial processes.
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Iodoacetamide by Merck Group
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Iodoacetamide is a chemical compound commonly used in biochemistry and molecular biology laboratories. It is a reactive compound that selectively modifies cysteine residues in proteins, thereby allowing for the study of protein structure and function. Iodoacetamide is often used in sample preparation procedures for mass spectrometry and other analytical techniques.
7
Strata x c18 spe column by Phenomenex
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The Strata X C18 SPE column is a solid phase extraction (SPE) product designed for sample preparation. It utilizes a reversed-phase C18 sorbent material to extract and purify analytes from various sample matrices.
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Bca protein assay kit by Bio-Rad
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The BCA protein assay kit is a colorimetric detection and quantitation method for determining total protein concentration in a sample. The kit uses bicinchoninic acid (BCA) to measure the reduction of copper ions by proteins in an alkaline environment, producing a purple-colored complex that can be measured spectrophotometrically.
9
Trypsin by Merck Group
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Trypsin is a serine protease enzyme that is commonly used in cell biology and biochemistry laboratories. Its primary function is to facilitate the dissociation and disaggregation of adherent cells, allowing for the passive release of cells from a surface or substrate. Trypsin is widely utilized in various cell culture applications, such as subculturing and passaging of adherent cell lines.
10
Q exactive mass spectrometer by Thermo Fisher Scientific
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The Q Exactive mass spectrometer is a high-resolution, accurate-mass (HRAM) instrument designed for advanced proteomics, metabolomics, and small molecule applications. It combines a quadrupole mass filter with a high-field Orbitrap mass analyzer to provide precise mass measurements and high-quality data.

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