Fast Top-Down Intact Protein Characterization with Capillary Zone Electrophoresis–Electrospray Ionization Tandem Mass Spectrometry

Capillary zone electrophoresis (CZE)–electrospray ionization tandem mass spectrometry (ESI-MS/MS) was applied for rapid top-down intact protein characterization. A mixture containing four model proteins (cytochrome <i>c</i>, myoglobin, bovine serum albumin (BSA), and β-casein) was used as the sample. The CZE–ESI-MS system was first evaluated with the mixture. The four model proteins and five impurities were baseline-separated within 12 min. The limits of detection [signal-to-noise ratio (S/N) = 3] of the four model proteins ranged from 20 (cytochrome <i>c</i>) to 800 amol (BSA). The relative standard deviations of migration time and intensity for the four model proteins were less than 3% and 30%, respectively, in quintuplicate runs. CZE–ESI-MS/MS was then applied for top-down characterization of the mixture. Three of the model proteins (all except BSA) and an impurity (bovine transthyretin) were confidently identified by database searching of the acquired tandem spectra from protein fragmentation. Modifications including phosphorylation, N-terminal acetylation, and heme group binding were identified

Fast Top-Down Intact Protein Characterization with Capillary Zone Electrophoresis–Electrospray Ionization Tandem Mass Spectrometry

Capillary zone electrophoresis (CZE)–electrospray ionization tandem mass spectrometry (ESI-MS/MS) was applied for rapid top-down intact protein characterization. A mixture containing four model proteins (cytochrome <i>c</i>, myoglobin, bovine serum albumin (BSA), and β-casein) was used as the sample. The CZE–ESI-MS system was first evaluated with the mixture. The four model proteins and five impurities were baseline-separated within 12 min. The limits of detection [signal-to-noise ratio (S/N) = 3] of the four model proteins ranged from 20 (cytochrome <i>c</i>) to 800 amol (BSA). The relative standard deviations of migration time and intensity for the four model proteins were less than 3% and 30%, respectively, in quintuplicate runs. CZE–ESI-MS/MS was then applied for top-down characterization of the mixture. Three of the model proteins (all except BSA) and an impurity (bovine transthyretin) were confidently identified by database searching of the acquired tandem spectra from protein fragmentation. Modifications including phosphorylation, N-terminal acetylation, and heme group binding were identified

Capillary Zone Electrophoresis–Electrospray Ionization-Tandem Mass Spectrometry for Top-Down Characterization of the <i>Mycobacterium marinum</i> Secretome

Capillary zone electrophoresis (CZE) with an electrokinetically pumped sheath-flow nanospray interface was coupled with a high-resolution Q-Exactive mass spectrometer for the analysis of culture filtrates from <i>Mycobacterium marinum</i>. We confidently identified 22 gene products from the wildtype <i>M. marinum</i> secretome in a single CZE–tandem mass spectrometry (MS/MS) run. A total of 58 proteoforms were observed with post-translational modifications including signal peptide removal, N-terminal methionine excision, and acetylation. The conductivities of aqueous acetic acid and formic acid solutions were measured from 0.1% to 100% concentration (v/v). Acetic acid (70%) provided lower conductivity than 0.25% formic acid and was evaluated as low ionic-strength and a CZE–MS compatible sample buffer with good protein solubility

Quantitative determination of peptides using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

A method is described for the quantitative determination of peptides using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Known limitations imposed by crystal heterogeneity, peptide ionization differences, data handling, and protein quantification with MALDI-TOF mass spectrometry are addressed in this method with a “seed crystal” protocol for analyte-matrix formation, the use of internal protein standards, and a software package called maldi_quant. The seed crystal protocol, a new variation of the fast-evaporation method, minimizes crystal heterogeneity and allows for consistent collection of protein spectra. The software maldi_quant permits rapid and automated analysis of peak intensity data, normalization of peak intensities to internal standards, and peak intensity deconvolution and estimation for vicinal peaks. Using insulin proteins in a background of other unrelated peptides, this method shows an overall coefficient of variance of 4.4%, and a quantitative working range of 0.58-37.5 ng bovine insulin per spot. Coupling of this methodology to powerful analytical procedures such as immunoprecipitation is likely to lead to the rapid and reliable quantification of biologically relevant proteins and their closely related variants

Capillary Zone Electrophoresis–Electrospray Ionization-Tandem Mass Spectrometry for Top-Down Characterization of the <i>Mycobacterium marinum</i> Secretome

Capillary zone electrophoresis (CZE) with an electrokinetically pumped sheath-flow nanospray interface was coupled with a high-resolution Q-Exactive mass spectrometer for the analysis of culture filtrates from <i>Mycobacterium marinum</i>. We confidently identified 22 gene products from the wildtype <i>M. marinum</i> secretome in a single CZE–tandem mass spectrometry (MS/MS) run. A total of 58 proteoforms were observed with post-translational modifications including signal peptide removal, N-terminal methionine excision, and acetylation. The conductivities of aqueous acetic acid and formic acid solutions were measured from 0.1% to 100% concentration (v/v). Acetic acid (70%) provided lower conductivity than 0.25% formic acid and was evaluated as low ionic-strength and a CZE–MS compatible sample buffer with good protein solubility

The eIF2 kinase GCN2 directs keratinocyte collective cell migration during wound healing via coordination of reactive oxygen species and amino acids

Healing of cutaneous wounds requires the collective migration of epithelial keratinocytes to seal the wound bed from the environment. However, the signaling events that coordinate this collective migration are unclear. In this report, we address the role of phosphorylation of eukaryotic initiation factor 2 (eIF2) and attendant gene expression during wound healing. Wounding of human keratinocyte monolayers in vitro led to the rapid activation of the eIF2 kinase GCN2. We determined that deletion or pharmacological inhibition of GCN2 significantly delayed collective cell migration and wound closure. Global transcriptomic, biochemical, and cellular analyses indicated that GCN2 is necessary for maintenance of intracellular free amino acids, particularly cysteine, as well as coordination of RAC1-GTP-driven reactive oxygen species (ROS) generation, lamellipodia formation, and focal adhesion dynamics following keratinocyte wounding. In vivo experiments using mice deficient for GCN2 validated the role of the eIF2 kinase during wound healing in intact skin. These results indicate that GCN2 is critical for appropriate induction of collective cell migration and plays a critical role in coordinating the re-epithelialization of cutaneous wounds