Advances in LC–MS/MS-based glycoproteomics: Getting closer to system-wide site-specific mapping of the N- and O-glycoproteome

Site-specific structural characterization of glycoproteins is important for understanding the exact functional relevance of protein glycosylation. Resulting partly from the multiple layers of structural complexity of the attached glycans, the system-wide site-specific characterization of protein glycosylation, defined as glycoproteomics, is still far from trivial leaving the N- and O-linked glycoproteomes significantly under-defined. However, recent years have seen significant advances in glycoproteomics driven, in part, by the developments of dedicated workflows and efficient sample preparation, including glycopeptide enrichment and prefractionation. In addition, glycoproteomics has benefitted from the continuous performance enhancement and more intelligent use of liquid chromatography and tandem mass spectrometry (LC–MS/MS) instrumentation and a wider selection of specialized software tackling the unique challenges of glycoproteomics data. Together these advances promise more streamlined N- and O-linked glycoproteome analysis. Tangible examples include system-wide glycoproteomics studies detecting thousands of intact glycopeptides from hundreds of glycoproteins from diverse biological samples. With a strict focus on the system-wide site-specific analysis of protein N- and O-linked glycosylation, we review the recent advances in LC–MS/MS based glycoproteomics. The review opens with a more general discussion of experimental designs in glycoproteomics and sample preparation prior to LC–MS/MS based data acquisition. Although many challenges still remain, it becomes clear that glycoproteomics, one of the last frontiers in proteomics, is gradually maturing enabling a wider spectrum of researchers to access this new emerging research discipline. The next milestone in analytical glycobiology is being reached allowing the glycoscientist to address the functional importance of protein glycosylation in a system-wide yet protein-specific manner.     

Strong humoral immune responses against SARS-CoV-2 Spike after BNT162b2 mRNA vaccination with a 16-week interval between doses

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Administration-Time Differences in the Pharmacokinetics of Gentamicin Intravenously Delivered to Human Beings

Administration-time differences of gentamicin pharmacokinetics were studied by crossover design after a single intravenous administration of gentamicin (80 mg) to 10 human subjects at 09:00 (morning time) and 22:00 (nighttime). The profiles of serum gentamicin concentration showed a significant statistical difference between 09:00 and 22:00, suggesting circadian variations of pharmacokinetic behaviors. A significant circadian rhythm of pharmacokinetic parameters as a function of time of day was noted in human subjects, showing lower total body clearance Cl_t and higher serum area under the curve (AUC) when given at nighttime. The half-life t_1/2 was shorter in the morning (2.82h ± 0.43h) when compared to the nighttime (2.97h ± 0.36h), but the difference was not statistically significant. The AUC was significantly greater in the morning (23.4 ± 3.84 μg-h/mL) than that in the nighttime (26.3 ± 5.79 μg-h/mL) (p<. 05), most likely because the Cl_t, was significantly higher when gentamicin was given in the morning (3.51 ± 0.57 L/h) versus in the nighttime (3.18 ± 0.65 L/h). Although the volume of distribution V_d decreased when given at nighttime, it was independent of the dosing time. From this study, there was an administration-time difference of gentamicin pharmacokinetics in human beings. The optimized dosing regimen of gentamicin can be decided by considering circadian rhythm and rest-activity routine so that minimized toxicity and effective therapy are established for patients. The current findings also can be applied to other drugs with circadian rhythms of pharmacokinetics and narrow therapeutic windows in clinical chronotherapeutics.     

Proteome and phosphoproteome profiling of non-small cell lung cancer cell line A549 treated with TRAIL

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is recognized for its promising therapeutic effects against cancer. However, mechanisms underlying the effect of TRAIL on protein expression, signal transduction, and apoptosis induction remain unclear. We surmised that a systematic analysis of the proteome and phosphoproteome associated with TRAIL signaling may help elucidate the mechanisms involved and facilitate the development of therapeutics. Therefore, we investigated the proteome and phosphoproteome of non-small cell lung cancer cell line A549 treated with TRAIL. Our results indicated that 126 proteins and 1684 phosphosites were markedly differentially expressed between the phosphate-buffered saline- and TRAIL-treated groups. The expression at protein and phosphosite levels were not completely consistent. Gene ontology functional analysis revealed that metal ion (zinc) binding was highly affected by TRAIL treatment. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed that almost all pathways that involved differentially expressed phosphosites were associated with apoptosis. We also identified an important kinase, AKT1, and its series of substrates in TRAIL signaling. The results of this study may provide guidance for future research on tumor therapy using TRAIL.     

阿扎胞苷单药或联合HAG方案在骨髓增生异常综合征中的临床观察

摘要 目的：探究阿扎胞苷（Azacitidine，AZA）单药或联合HAG方案治疗骨髓增生异常综合征（Myelodysplastic syndromes，MDS）的临床疗效及安全性。方法：回顾性分析49例MDS患者的临床资料，根据治疗方法不同分为支持治疗组和含AZA组（单药或联合HAG方案），统计分析患者的临床疗效及不良反应情况。结果：支持治疗组的总有效率（Overall response rate，ORR）为30.00 %（6/20），包括0例完全缓解（Complete remission，CR），1例骨髓完全缓解（Marrow complete remission，mCR），2例部分缓解（Partial remission，PR）, 3例血液学改善（Hematological improvement，HI）。含AZA组的ORR为65.52 %，包括8例CR、2例mCR、4例PR、5例HI。其中单药组的ORR为46.67 %, 包括2例CR、1例mCR、2例PR、2例HI；联合组的ORR为85.71 %，包括6例CR、1例mCR、2例PR、3例HI。与支持治疗组相比，含AZA组的完全缓解率（CR+mCR）及ORR显著增高，差异有统计学意义（P＜0.05）。患者最常见的不良反应是III-IV级骨髓抑制（18/29）及继发感染（10/29），且随着疗程数的增加不良事件逐渐减少。含AZA组患者的中位总生存（Overall survival，OS）时间及中位无进展生存时间（Progression-free survival，PFS）时间显著延长（P＜0.05）。结论：该小系列研究的初步结果表明，与支持治疗相比，AZA单药或联合HAG方案治疗MDS有更高的治疗反应，可延长患者总生存期，患者有良好的耐受性，且联合治疗方案可能有更好的疗效。     

Transfusion regimen in beta thalassemia major using magnetic measurement techniques

The magnetic behaviour of erythrocytes from patients suffering from beta thalassemia major was investigated. All samples exhibited diamagnetic behaviour. Measurements were made before and after blood transfusion treatment. We suggest that the amount of blood given to the patient should be controlled in a way that the diamagnetic susceptibility after blood transfusion should be equal to that for the normal erythrocytes. In this way a specific range of haemoglobin level (Hgb) should be maintained when the patient is subjected to long-term hypertransfusional treatment.     

Integration of data-independent acquisition (DIA) with co-fractionation mass spectrometry (CF-MS) to enhance interactome mapping capabilities

Proteomics technologies are continually advancing, providing opportunities to develop stronger and more robust protein interaction networks (PINs). In part, this is due to the ever-growing number of high-throughput proteomics methods that are available. This review discusses how data-independent acquisition (DIA) and co-fractionation mass spectrometry (CF-MS) can be integrated to enhance interactome mapping abilities. Furthermore, integrating these two techniques can improve data quality and network generation through extended protein coverage, less missing data, and reduced noise. CF-DIA-MS shows promise in expanding our knowledge of interactomes, notably for non-model organisms (NMOs). CF-MS is a valuable technique on its own, but upon the integration of DIA, the potential to develop robust PINs increases, offering a unique approach for researchers to gain an in-depth understanding into the dynamics of numerous biological processes.