Quantitative Phosphoproteomics Requires the Amount of Peptides
In quantitative phosphoproteomics analysis, peptide abundance is a critical factor, as it directly impacts the sensitivity, reproducibility, and quantitative accuracy of the experiments. The following are key considerations and suggestions:
Sample Preparation
1. Protein Extraction
Extract total proteins from cell or tissue samples, typically requiring several milligrams.
2. Protein Digestion
Using proteolytic enzymes like trypsin, digest the proteins into peptides. Generally, 1-5 mg of protein can yield sufficient peptides for subsequent analyses.
Peptide Quantity
1. Initial Sample Quantity
Depending on the complexity of phosphoproteins and the sample, the initial sample typically ranges from 1-5 mg.
2. Peptide Amount
For LC-MS/MS analysis, 0.1-2 µg of peptides are usually required. More complex samples may necessitate a higher peptide quantity to ensure comprehensive detection of phosphorylated residues.
Phosphopeptide Enrichment
1. Enrichment Strategy
Phosphopeptides, being a minority in complex mixtures, require enrichment using strategies such as metal oxide affinity chromatography (MOAC), immunoprecipitation, or titanium dioxide (TiO2).
2. Sample Quantity Pre- and Post-Enrichment
Before enrichment, 100-500 µg of peptides is needed; post-enrichment yields about 1-10 µg of phosphopeptides for LC-MS/MS analysis.
Quantitative Analysis
1. Quantification Methods
Popular methodologies include isotope labeling (e.g., SILAC, TMT, iTRAQ) or label-free quantification.
2. Required Peptide Amount
Regardless of quantification strategy, typically 0.5-2 µg of peptides are injected into the LC-MS/MS system for analysis.
In conclusion, quantitative phosphoproteomics typically involves extracting 1-5 mg of total protein, followed by digestion and enrichment resulting in the analysis of 0.1-2 µg of peptides via LC-MS/MS. The specific quantities depend on sample complexity, mass spectrometer sensitivity, and quantification technique.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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