Search

In this work, 193 nm ultraviolet photodissociation-mass spectrometry (UVPD-MS) was applied to investigate supercharging reagent sulfolane in protein sequence and structure characterization. All datasets were collected by using an Orbitrap MS system equipped with an ArF 193 nm EX50 excimer Laser. The 193 nm UV laser has the advantages of high brightness, good stability, and propagate in air, which can directly excite and dissociate the protein backbone within 5 ns (single pulse). Previous reports demonstrated that the partial protein high-order structures and non-covalent interactions could be retained in the UVPD fragments and the site-specific fragmentation yields (FYs) revealed the dynamic protein structure alterations under different conditions. Therefore, the combination of UVPD with nMS provides a new research paradigm for the characterization of protein structure changes. In this work, we further demonstrated 193 nm UVPD was superior to high-energy collision induced dissociation (HCD) in fragmenting and sequencing the non-denatured carbonic anhydrase II (CA) generated in native electrospray ionization (ESI). UVPD could efficiently activate and fragmentate the non-denatured CA with different charge states in just 5 ns, achieving cleavage sequence coverage 79.8% for CA Z10. Then, 0.25% supercharging reagent sulfolane was added into the protein solution and the CA charge distribution was significantly moved to higher charge states under identical native ESI conditions. However, higher-order structures (HOS) and non-covalent interactions could be obtained as the CA charge distribution was still concentrated and significantly different from the spectra features of denatured CA. The UVPD cleavage sequence coverage of the CA was increased to 87.2%, including the regions that were difficult to be dissociated and characterized without sulfolane addition. In addition, the non-covalent of CA and Zn2+ remained stable, indicating that the addition of sulfoxide had little influence on the Zn2+ binding region. The UVPD sequence coverage of CA Z11 was increased by 7.4% after the addition of sulfoxide, but the site FYs were almost not changed, demonstrating the corresponding local structures were nearly not changed. However, the higher charge states introduced by sulfolane also exhibited the risk of inducing specific structure alteration of part of protein regions. In conclusion, it was infered that supercharging reagent is efficient in modulating the charge state distribution of proteins in native ESI-MS characterization and can expand the MS detection mass range. The introduction of supercharging reagent can further improve the UVPD sequence coverage, including the structure-compact regions hard to be fragmented. Although part of the protein HOS and non-covalent interactions seems retained, the influence of higher charge states on protein structures should be considered in dynamic protein structure investigation. [ABSTRACT FROM AUTHOR]

Protein related tumor biomarkers have been widely used in clinically assisted diagnosis and disease surveillance because they can indicate the physiological state, pathological process and post-drug reaction of cancer patients. Therefore, it is of great significance to accurately measure tumor biomarkers. However, due to the wide dynamic range of body fluid, the existence of high abundant proteins and diverse post-translation modifications pose a great challenge to the accurate quantification of protein related tumor biomarkers. Liquid chromatography-mass spectrometry (LC-MS) combines the high-efficiency separation capability of chromatography technology and the high sensitivity and high throughput detection capability of mass spectrometry technology, which has become a powerful tool for the discovery, verification and analysis of biomarkers. Currently, LC-MS has been widely applied in the field of quantitative research on protein related tumor biomarkers. In this paper, research progress in the quantitative methods for protein related tumor biomarkers in recent years was summarized from the sample pretreatment approach and mass spectrometry scanning strategy. In complex biological samples, protein related tumor biomarkers are usually at ultra-low expression level, which make it difficult to directly analyze protein related tumor biomarkers by mass spectrometry. Therefore, the development of appropriate sample pretreatment methods (e.g. high abundance protein removal, target protein enrichment, quantitative peptide collection, isotope labelling, etc.) to reduce the complexity of biological samples before quantification analysis is a key step to ensure accurate measurement results. In addition, primary MS analysis can only provide limited biological information, which can not effectively distinguish the substances with similar mass-to-charge ratio, and can not meet the needs of protein related tumor biomarkers quantification. Thus, multi-stage mass spectrometry scanning strategy (such as multiple reaction monitoring, parallel reaction monitoring, etc.) has become an effective method for protein quantitative analysis. Although the rapid development of mass spectrometry technology in resolution, mass accuracy and analysis speed provides opportunities for accurate protein analysis, it is still difficult to accurately quantify low-abundant proteins for the high complexity of biological samples in body fluid. Moreover, due to the limitation of the automation for both mass spectrometer device and sample pretreatment, quantitative analysis of protein related tumor biomarkers based on LC-MS is still not applied to the clinical detection on a large scale. Therefore, it is necessary to continuously develop efficient and rapid sample pretreatment approaches, accurate and reliable mass spectrometry scanning strategies and the easy automation technology. In the future, more reliable detection methods for protein biomarkers may make disease screening, diagnosis and treatment more precise and more efficient. [ABSTRACT FROM AUTHOR]

Disulfide bond in protein is an important post-translational modification and it is extremely essential for stabilizing the three-dimensional structure of proteins, maintaining the correct folded conformation and regulating their biological activities. Therefore, disulfide bonds assignment is of importance to understand life process and to develop protein pharmaceutical. Mass spectrometry technology can serve not only as a means cleaving the disulfide bonds, but also as an analysis tool assigning disulfide bonds connectivity. As a fragmentation means, mass spectrometry has various of MS/MS fragmentation modes. As a detector, it has the advantages of high sensitivity and high accuracy for mass spectrometry information. With these merits, mass spectrometry could fulfill the desire in the characterization of disulfide bonds. This paper summarized the mass spectrometric methods for locating disulfide bonds in proteins or peptides, according to the medium where the disulfide bonds were broken and the cleavage mechanism. Two cleavage mediums-(solution and gas phase) were divided. According to the cleavage mechanism, reduction/oxidation reaction using chemical reagents, electrochemical reduction based on electrode reaction, and post-column reduction were classified, in which disulfide bonds were cleaved in solution and the mass spectrometer was mainly used as an analysis tool. Meanwhile, molecular collision fragmentation including CID and HCD, electrons capture fragmentation (ECD and ETD), photons absorption fragmentation (e.g. MALDI-ISD and UVPD), and cleavage based on ion-ion reactions were summarized, in which disulfide bonds were cleaved in gas phase. For these methods, mass spectrometer was used not only as an analysis tool but also more importantly as a cleavage approach. This review included most disulfide bonds assignment methods and would facilitate bioinformatics-related researchers to choose an appropriate mass spectrometric method. Additionally, at the end of this paper, several new challenges in the field of disulfide bond assignment were put forward such as the characterization of multiple disulfide bonds in a short peptide, insufficient study of the disulfide bond artifacts, and the new methods based on novel instruments like UVPD. Moreover, in terms of methodological study, it would be promoted by the development of the new ionization and fragmentation technique producing new kinds of characteristic ions and corresponding data processing tools emerged in the future. Through this review, it was expected to develop more effective mass spectrometric methods for disulfide bonds characterization. [ABSTRACT FROM AUTHOR]

The objective of the present study was to study the proteins and peptides of goat horn (Caprae Hircus Cornu, GH) based on the shotgun strategy. By using LTQ/Orbitrap MS, proteins and peptides were identified from GH aqueous extract. In total, 52 proteins were identified from GH fraction with the molecular weight larger than 3 ku, and 1288 peptides were identified from GH fraction with the molecular weight lower than 3 ku, and these peptides were hydrolyzed from 46 proteins. The identified proteins were keratin, collagen, keratin-associated protein, etc. It can be speculated that the particular “hot regions” in the amino acid sequence which were easier to proteolysis, drop, dissolve and generate a number of peptides. According to the present strategy, proteins and peptides derived from GH aqueous extract can be rapid and efficient identified. The strategy can be applied in analysis and identification of proteins in animal horn derived TCMs, and provide a reference for investigate the material basis of animal horn derived TCMs. [ABSTRACT FROM AUTHOR]