Computational protocol: An Evaluation of Blood Compatibility of Silver Nanoparticles

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Protocol publication

[…] Approximately 0.5–1 μg of peptides were loaded for 80 min gradients, respectively. Peptides were separated on an 20 cm reversed phase capillary emitter column (inner diameter 100 μm, 5 μm Venusil XBP C18 resin (Agela Technologies, China)) and analyzed on the Q Exactive instrument (Thermo Fisher Scientific). Peptides were loaded in 0.2% (v/v) formic acid solution and eluted with a nonlinear 80 min gradient of 5–30% buffer B (0.1% (v/v) formic acid, 90% (v/v) acetonitrile) at a flow rate of 300 nL/min. MS data were acquired by Thermo X calibur (2.0) with an data-dependent MS/MS scans (TopN = 15). Target value for the full MS scan was 3 × 106 in the 320–1,700 m/z range with a maximum injection time of 20 ms and a resolution of 70,000 at m/z of 200. Isolation window was 1.5 m/z and normalized collision energy of 27. MS/MS scans resolution was 17,500 at m/z 200 with an ion target value of 1 × 105 and a maximum injection time of 60 ms. To avoid peptide’s repeated sequencing, exclusion time was set to 60 s.MS raw files were processed with MaxQuant software (version 1.4.1.2) with label free quantification work flow (MaxLFQ). The peak lists were searched against the human Uniprot FASTA (version 20 April 2014 (88725 entries)) with reversed protein sequences and a common contaminants database (247 entries) by Andromeda search engine. The search included cysteine carbamidomethylation and variable modifications of methionine oxidation and protein N-terminal acetylation. Enzyme specificity was set as C-terminal to arginine and lysine, and a maximum of two missed cleavages were allowed in the database search. Peptides with at least six amino acids were considered for identification. The false discovery rate for both peptides and proteins was set at 1%. [...] Data analysis was performed with Perseus software in the MaxQuant computational platform and by R statistical computing environment. The three technological repeats of each sample were set as one experiment when running MaxLFQ process, and the average value (Av.) of biological repetition of each identified protein’s LFQ intensity was used in following analysis. Gene ontology (GO) biological process of proteins and their interactions network were performed by Cytoscape plugged ClueGO + CluePedia with P-values (Bonferroni step down correction) <0.05. The concentrations of plasma proteins were derived from Plasma Proteome Database (PPD; http://www.plasmaproteomedatabase.org/). For correlation analysis of corona proteins’ pI and MW with their LFQ intensity, two tailed Spearman test was used. […]

Pipeline specifications

Software tools MaxQuant, Andromeda, Perseus, ClueGO, CluePedia
Databases PPD
Application MS-based untargeted proteomics
Diseases Blood Platelet Disorders, Thrombosis
Chemicals Povidone, Silver