Functional Microgels Assisted Tryptic Digestion and Quantification of Cytochrome c Through Internal Standard Mass Spectrometry

Abstract

Quantitation of cytochrome c (Cyt c) in cell lysates through surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) using gold nanoparticles (Au NPs) as the matrix and GR-10 peptide as an internal standard has been demonstrated. To shorten digestion time, temperature sensitive microgels containing trypsin (TR) and Au NPs have been employed. As-prepared functional microgels (TR/Au NPs/MGs) allow digestion of Cyt c within 15 s under microwave irradiation. The internal standard SALDI-MS approach provides linearity (R(2) = 0.98) of MS signal ratio (I 1168.6/I 1067.6) of the tryptic digested peptide (m/z 1168.6) to GR-10 peptide (m/z 1067.6) against the concentration of Cyt c ranging from 25 to 200 nM, with a limit of detection (at a signal-to-noise ratio of 3) of 10 nM. This approach has been validated by the analysis of the lysates of HeLa cells, with an average concentration of 13.7 ± 3.5 μM for cytoplasmic Cyt c. Increased concentrations of Cyt c in the HeLa cells treated with etoposide (a commercial drug) or carbon dots (potential drug) have been revealed through this simple, sensitive, and rapid SALDI-MS approach, supporting the drugs induced Cyt c-mediated apoptosis of the cells. This study has shown that this internal standard SALDI-MS approach holds great potential for cell study.

Thermoresponsive Arrays Patterned via Photoclick Chemistry: Smart MALDI Plate for Protein Digest Enrichment, Desalting, and Direct MS Analysis

“…In comparison, same amounts of cytochrome c and neurotensin were loaded on the 1 H ,1 H ,2 H ,2 H -perfluorodecanethiol-modified ITO substrate and 280 nm absorption peaks were clearly observed from the 1st and 2nd time rinsing solutions, indicating a sample loss to some extent from a non-thermo-responsive hydrophobic 2D surface (Figure S11c,d). α-Casein is then introduced as the internal standard for quantifying the amount of cytochrome c retained on the PNIPAM patterns after the on-plate pretreatment process . 1 mg/mL α-casein was added into 1 mg/mL cytochrome c aqueous solution in the absence of salt as the standard solution.…”

“…α-Casein is then introduced as the internal standard for quantifying the amount of cytochrome c retained on the PNIPAM patterns after the on-plate pretreatment process. 52 1 mg/mL α-casein was added into 1 mg/mL cytochrome c aqueous solution in the absence of salt as the standard solution. The mixture solution was cocrystallized with CHCA on the smart MALDI plate, and the internal standard/analyte (i/a) ratios were determined as 1.70 by comparing the intensities of the internal standard peak [αcasein] 2+ at m/z 11 866 and analyte peak [cyt-c] + at m/z 12 320 (Figure S12a).…”

Development of Biomimetic Antimicrobial Platelet-Like Particles Comprised of Microgel Nanogold Composites

“…Microgels were lyophilized and then rehydrated at a microgel concentration of 10 mg/mL with a 0.05 mg/mL aqueous suspension of gold nanospheres (5, 50, or 100 nm diameter) in 2 mM sodium citrate (nanoComposix, San Diego, CA) with shaking at room temperature overnight. The second method of NGC fabrication utilized a covalent nanogold composite (cNGC) fabrication method in which gold-microgel composites were synthesized in a twostep procedure modified from Chen et al [27]. Briefly, tetra(hydroxymethyl)phosphonium chloride (THPC)-mediated reduction of HAuCl 4 -3H 2 O was used to form small Au NPs, which were then grown in size using hydroxylamine and Au 3+ solution.…”