The peptide and phospho-peptide samples were both analyzed using a nanoLC system (Waters NanoAcquity LC, Waters Corporation) coupled to a Q ExactiveTM Hybrid Quadrupole-OrbitrapTM Mass Spectrometer (Thermo Fisher Scientific) in data dependent mode. All mass spectrometry data were searched using MS-GF+ (Kim et al., 2008 (link); Kim and Pevzner, 2014 (link)) to identify peptides by scoring MS/MS spectra against peptides derived from the whole protein sequence database. The MS-GF + results were filtered based on 1% false discovery rate (FDR) and less than 5-ppm mass accuracy. Based on spectral count MS data were considered only those peptides that were identified in at least 2 out of 4 experimental replicates with a positive spectrum, independent of the spectral count value. The phospho-peptide results were also searched using AScore algorithm (Gerber et al., 2006 (link)) to provide a probability-based score for each identified phospho-peptide. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (Perez-Riverol et al., 2019 (link)) with the dataset identifier PXD013964 and 10.6019/PXD013964.
Waters nanoacquity lc
Manufactured by Waters Corporation
The Waters NanoAcquity LC is a liquid chromatography system designed for high-performance, low-flow applications. It features precise flow control, compatibility with a variety of column sizes, and advanced detection capabilities.
Automatically generated - may contain errors
3 protocols using waters nanoacquity lc
1
Peptide and Phospho-Peptide Mass Spectrometry Workflow
2
Phosphoproteome Analysis of Protein Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were reduced with 10 mM dithiothreitol for 1 h at 56 °C and subsequently alkylated with 55 mM iodoacetamide. Samples were digested with trypsin at 1:20 enzyme-to-substrate ratio. Digested samples were desalted using C18 solid phase extraction tubes. The resulting peptide samples were concentrated and a BCA assay was performed to determine the peptide concentration and samples were diluted with nanopure water for MS analysis. Desalted peptides were labeled with 8-plex iTRAQ reagents (AB SCIEX) according to the manufacturer’s instructions. Peptide aliquots for each sample (200 mg) were dried for TiO2 enrichment, and used for phosphoproteome analysis; TiO2 enrichment of phosphopeptides followed a previously established protocol [102 (link)]. Phosphopeptide samples were analyzed using a nanoESI system (Waters NanoAcquity LC, Waters Corporation) coupled to a Q Exactive HF mass spectrometer (Thermo Fisher Scientific). Proteomics data were analyzed using a combination of Proteome Discoverer (v1.4) and Mascot (v2.3) software. The MS results were filtered based on a 5% false discovery rate and phosphoRS probability ≥ 0.75. Phosphopeptide abundance changes >1.5-fold were considered and subjected to downstream analysis.
3
Quantitative Phosphoproteome Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were reduced with 10 mM for 1 h at 56°C and subsequently alkylated with 55 mM iodoacetamide. Samples were digested with trypsin at 1:20 enzyme-to-substrate ratio. Digested samples were desalted using C18 solid phase extraction tubes. The resulting peptide samples were concentrated and a BCA assay was performed to determine the peptide concentration and samples were diluted with nanopure water for MS analysis. Desalted peptides were labeled with 8-plex iTRAQ reagents (AB SCIEX) according to the manufacturer's instructions. Peptide aliquots for each sample (200 mg) were dried for TiO 2 enrichment, and used for phosphoproteome analysis; TiO 2 enrichment of phosphopeptides followed a previously established protocol (102) . Phosphopeptide samples were analyzed using a nanoESI system (Waters NanoAcquity LC, Waters Corporation) coupled to a Q Exactive HF mass spectrometer (Thermo Fisher Scienti c). Proteomics data were analyzed using a combination of Proteome Discoverer (v1.4) and Mascot (v2.3) software. The MS results were ltered based on a 5% false discovery rate and phosphoRS probability ≥ 0.75. Phosphopeptide abundance changes > 1.5-fold were considered and subjected to downstream analysis.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!