Top-down mass spectrometry of native proteoforms and their complexes: a community study.

The combination of native electrospray ionization with top-down fragmentation in mass spectrometry (MS) allows simultaneous determination of the stoichiometry of noncovalent complexes and identification of their component proteoforms and cofactors. Although this approach is powerful, both native MS and top-down MS are not yet well standardized, and only a limited number of laboratories regularly carry out this type of research. To address this challenge, the Consortium for Top-Down Proteomics initiated a study to develop and test protocols for native MS combined with top-down fragmentation of proteins and protein complexes across 11 instruments in nine laboratories. Here we report the summary of the outcomes to provide robust benchmarks and a valuable entry point for the scientific community.

Recent progress in the analysis of protein deamidation using mass spectrometry.

Deamidation is a nonenzymatic and spontaneous posttranslational modification (PTM) that introduces changes in both structure and charge of proteins, strongly associated with aging proteome instability and degenerative diseases. Deamidation is also a common PTM occurring in biopharmaceutical proteins, representing a major cause of degradation. Therefore, characterization of deamidation alongside its inter-related modifications, isomerization and racemization, is critically important to understand their roles in protein stability and diseases. Mass spectrometry (MS) has become an indispensable tool in site-specific identification of PTMs for proteomics and structural studies. In this review, we focus on the recent advances of MS analysis in protein deamidation. In particular, we provide an update on sample preparation, chromatographic separation, and MS technologies at multi-level scales, for accurate and reliable characterization of protein deamidation in both simple and complex biological samples, yielding important new insight on how deamidation together with isomerization and racemization occurs. These technological progresses will lead to a better understanding of how deamidation contributes to the pathology of aging and other degenerative diseases and the development of biopharmaceutical drugs.

A New Real-Time Pinch Detection Algorithm Based on Model Reference Kalman Prediction and SRMS for Electric Adjustable Desk.

This paper presents a new algorithm based on model reference Kalman torque prediction algorithm combined with the sliding root mean square (SRMS). It is necessary to improve the accuracy and reliability of the pinch detection for avoiding collision with the height adjustable desk and accidents on users. Motors need to regulate their position and speed during the operation using different voltage by PWM (Pulse Width Modulation) to meet the requirement of position synchronization. It causes much noise and coupling information in the current sampling signal. Firstly, to analyze the working principle of an electric height adjustable desk control system, a system model is established with consideration of the DC (Direct Current) motor characteristics and the coupling of the system. Secondly, to precisely identify the load situation, a new model reference Kalman perdition method is proposed. The load torque signal is selected as a pinch state variable of the filter by comparing the current signal. Thirdly, to meet the need of the different loads of the electric table, the sliding root means square (SRMS) of the torque is proposed to be the criterion for threshold detection. Finally, to verify the effectiveness of the algorithm, the experiments are carried out in the actual system. Experimental results show that the algorithm proposed in this paper can detect the pinched state accurately under different load conditions.

Native top-down mass spectrometry for monitoring the rapid chymotrypsin catalyzed hydrolysis reaction.

Enzymes play crucial roles in life sciences, pharmaceuticals and industries as biological catalysts that speed up biochemical reactions in living organisms. New catalytic reactions are continuously developed by enzymatic engineering to meet industrial needs, which thereby drives the development of analytical approaches for real-time reaction monitoring to reveal catalytic processes. Here, taking the hydrolase- chymotrypsin as a model system, we proposed a convenient method for monitoring catalytic processes through native top-down mass spectrometry (native TDMS). The chymotrypsin sample heterogeneity was first explored. By altering sample introduction modes and pHs, covalent and noncovalent enzymatic complexes, substrates and products can be monitored during the catalysis and further confirmed by tandem MS. Our results demonstrated that native TDMS based catalysis monitoring has distinctive strength on real-time inspection and continuous observation, making it a promising tool for characterizing more biocatalysts.

Top-down mass spectrometry of native proteoforms and their complexes: A community study.

The combination of native electrospray ionisation with top-down fragmentation in mass spectrometry allows simultaneous determination of the stoichiometry of noncovalent complexes and identification of their component proteoforms and co-factors. While this approach is powerful, both native mass spectrometry and top-down mass spectrometry are not yet well standardised, and only a limited number of laboratories regularly carry out this type of research. To address this challenge, the Consortium for Top-Down Proteomics (CTDP) initiated a study to develop and test protocols for native mass spectrometry combined with top-down fragmentation of proteins and protein complexes across eleven instruments in nine laboratories. The outcomes are summarised in this report to provide robust benchmarks and a valuable entry point for the scientific community.