Capillary zone electrophoresis-tandem mass spectrometry with activated ion electron transfer dissociation for large-scale top-down proteomics

Capillary zone electrophoresis (CZE)-tandem mass spectrometry (MS/MS) has been recognized as an efficient approach for top-down proteomics recently for its high-capacity separation and highly sensitive detection of proteoforms. However, the commonly used collision-based dissociation methods often cannot provide extensive fragmentation of proteoforms for thorough characterization. Activated ion electron transfer dissociation (AI-ETD), that combines infrared photoactivation concurrent with ETD, has shown better performance for proteoform fragmentation than higher energy-collisional dissociation (HCD) and standard ETD. Here, we present the first application of CZE-AI-ETD on an Orbitrap Fusion Lumos mass spectrometer for large-scale top-down proteomics of Escherichia coli (E. coli) cells. CZE-AI-ETD outperformed CZE-ETD regarding proteoform and protein identifications (IDs). CZE-AI-ETD reached comparable proteoform and protein IDs with CZE-HCD. CZE-AI-ETD tended to generate better expectation values (E values) of proteoforms than CZE-HCD and CZE-ETD, indicating a higher quality of MS/MS spectra from AI-ETD respecting the number of sequence-informative fragment ions generated. CZE-AI-ETD showed great reproducibility regarding the proteoform and protein IDs with relative standard deviations less than 4% and 2% (n = 3). Coupling size exclusion chromatography (SEC) to CZE-AI-ETD identified 3028 proteoforms and 387 proteins from E. coli cells with 1% spectrum level and 5% proteoform-level false discovery rates. The data represents the largest top-down proteomics dataset using the AI-ETD method so far. Single-shot CZE-AI-ETD of one SEC fraction identified 957 proteoforms and 253 proteins. N-terminal truncations, signal peptide cleavage, N-terminal methionine removal, and various post-translational modifications including protein N-terminal acetylation, methylation, S-thiolation, disulfide bonds, and lysine succinylation were detected

Users of ‘diet’ drinks who think that sweetness is calories

We present the first experiment that was based on a novel analysis of the mental processes of choice. Sensed material characteristics such as the sweetness of a drink and symbolic attributes such as the source of sweetness stated on the label are put into the same units of influence on the response. Most users of low-calorie drinks thought about the energy in a drink quite differently from the way they decided how sweet and how low in calories they liked the drink to be. Also the female diet drink users thought about energy content differently from most of the male users of sugar drinks. In both groups’ ratings of likelihood of choice and in sugar drink users’ estimates of energy content, sweetness and labelled calories were usually treated as separate stimuli or ideas. In contrast, some female diet drink users treated sweetness and perceived calories as the same, whereas no male sugar drink user did. Such findings illustrate how this approach spans the gap between sensory perception and conceptualised knowledge

Mean %PP of plasma amino acid and derivatives measured by CE-MS for lean healthy (LH, n = 10, white bars), metabolically healthy obese (MHO, n = 10, grey bars) and metabolically unhealthy obese (MUO, n = 10, black bars).

<p>Significant %PP amino acids between the three groups were identified using a non-parametric ANOVA Kruskal-Wallis (p < 0.05) test followed by a post-hoc Mann-Whitney test (p < 0.05). Data is presented as mean %PP ± SEM.</p

Associations between amino acids and derivatives, and fatty acids, with fasting and postprandial indices of insulin sensitivity.

<p>Significance was set at p ≤ 0.01 using Bonferroni correction for multiple testing. Linear regressions were adjusted for age, sex, and BMI.</p><p>Associations between amino acids and derivatives, and fatty acids, with fasting and postprandial indices of insulin sensitivity.</p