Structural conservation among variants of the SARS-CoV-2 spike postfusion bundle

Variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) challenge currently available COVID-19 vaccines and monoclonal antibody therapies due to structural and dynamic changes of the viral spike glycoprotein (S). The heptad repeat 1 (HR1) and heptad repeat 2 (HR2) domains of S drive virus–host membrane fusion by assembly into a six-helix bundle, resulting in delivery of viral RNA into the host cell. We surveyed mutations of currently reported SARS-CoV-2 variants and selected eight mutations, including Q954H, N969K, and L981F from the Omicron variant, in the postfusion HR1HR2 bundle for functional and structural studies. We designed a molecular scaffold to determine cryogenic electron microscopy (cryo-EM) structures of HR1HR2 at 2.2–3.8 Å resolution by linking the trimeric N termini of four HR1 fragments to four trimeric C termini of the Dps4 dodecamer from Nostoc punctiforme. This molecular scaffold enables efficient sample preparation and structure determination of the HR1HR2 bundle and its mutants by single-particle cryo-EM. Our structure of the wild-type HR1HR2 bundle resolves uncertainties in previously determined structures. The mutant structures reveal side-chain positions of the mutations and their primarily local effects on the interactions between HR1 and HR2. These mutations do not alter the global architecture of the postfusion HR1HR2 bundle, suggesting that the interfaces between HR1 and HR2 are good targets for developing antiviral inhibitors that should be efficacious against all known variants of SARS-CoV-2 to date. We also note that this work paves the way for similar studies in more distantly related viruses.

Three previously unknown beta-coronaviruses have emerged in the first two decades of this century: severe acute respiratory syndrome coronavirus (SARS-CoV) in 2003, Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in late 2019 (1). The most recent outbreak of SARS-CoV-2 that causes coronavirus disease 2019 (COVID-19) has claimed about 6 million lives in 2 y, and several variants of concern have emerged around the globe despite the relatively low mutation rate of coronaviruses (2). Some of these variants pose a challenge to currently available vaccines (3–6), likely due to structural changes of the target of these vaccines (7–11). Hence, there is an urgent need for new antiviral therapeutics (12) that target regions of viruses with conserved structural features that are less likely to be affected by mutations.SARS-CoV, MERS-CoV, and SARS-CoV-2 are enveloped viruses that rely on membrane fusion to deliver RNA to the host cell (13). In each case, the process of viral membrane fusion (14, 15) is mediated by the trimeric viral spike glycoprotein (S) that is cleaved into S1 and S2 subunits by multiple host proteases upon infection (16) (Fig. 1A). S1 recognizes the human angiotensin-converting enzyme 2 (ACE2) receptor and dissociates from S2. Subsequently, S2 undergoes substantial conformational changes that drive membrane remodeling. Similar to other enveloped viruses (14, 15), this process likely proceeds via an intermediate extended state that pulls together the two membranes via the transmembrane domain and fusion peptide of the S2 subunit (17). Two heptad repeat regions, HR1 and HR2, distant from each other in the prefusion S, drive membrane fusion by assembly into a six-helix bundle (18). This HR1HR2 bundle formation is thought to provide the energy for membrane fusion and is therefore a target for therapeutics, as exemplified by peptide inhibitors that disrupt infection by the HIV-1 (19, 20), SARS-CoV (21), MERS-CoV (22), SARS-CoV-2 (23–25), human parainfluenza virus 3 (26), and respiratory syncytial virus (26).Open in a separate windowFig. 1.Mutations of interest in the HR1HR2 bundle of SARS-CoV-2 variants. (A) Schematic diagram of the domain structures of the SARS-CoV-2 spike protein. The N and C termini are labeled on the left and right, respectively. FP, fusion peptide; HR1, heptad repeat 1; HR2, heptad repeat 2; TM, transmembrane region. (B) Locations of the five selected point mutations of SARS-CoV-2 variants (black spheres) and the three mutations of the SARS-CoV-2 Omicron variant (purple spheres) indicated in the crystal structure of the HR1HR2 bundle (PDB ID code 6lxt). Two HR2 residues, R1185 and N1187, that may be affected by the selected mutations are shown as red spheres. The HR1 and HR2 fragments are colored as light blue and light red, respectively. (C) Effects on fusion activity of these mutations. The fusion activity is shown as a percentage (Left)/fold change (Right) relative to that of the wild type (Materials and Methods). The Omicron construct used here for the fusion assay has three mutations—Q954H, N969K, and L981F—in the HR1 portion of the HR1HR2 bundle, but not other mutations from different regions of the spike found in the Omicron variant. *P < 0.05, **P < 0.01, ***P < 0.001, by a Student’s t test.Despite the established value of inhibitors targeting formation of the HR1HR2 bundle, the structural plasticity of this bundle upon mutation is largely unknown. Comparison with distantly related viruses suggests that the overall architecture is maintained despite vast differences in primary sequence (SI Appendix, Fig. S1). To what degree does the structure of the HR1HR2 bundle change upon mutation? To address this question, we surveyed mutations of all currently known variants (including Omicron) of SARS-CoV-2 S in the postfusion HR1HR2 bundle, selected eight mutations of potential interest, and investigated their effects on structure and function.Structural characterization of the HR1HR2 bundle has proven surprisingly challenging. To date, two successful approaches for determining structures of the HR1HR2 bundle have been employed. First, several HR1HR2 structures with the HR1 and HR2 domains synthetically linked were determined by X-ray crystallography (2.9 Å, Protein Data Bank [PDB] ID code 6lxt; 1.5 Å, PDB ID code 6m1v) (23, 25). Second, a sample of postfusion S2 was generated from a recombinant source (mammalian HEK-293F cells) expressing full-length S; as such, multiple states of S undergoing spontaneous transition from the prefusion to the postfusion state were present in the sample and the postfusion structure was determined by single-particle cryogenic electron microscopy (cryo-EM) (3.0 Å, PDB ID code 6xra) (18). Although the structures of the postfusion HR1HR2 bundle are similar, there are differences between these structures and the local resolution is quite variable or limited. More importantly, neither approach is particularly suited for efficient structure determination of multiple mutants at high resolution. Therefore, we decided to develop a platform for using single-particle cryo-EM to efficiently determine structures of HR1HR2 bundles at atomic resolution.The postfusion HR1HR2 bundle of SARS-CoV-2 is a 115 × 25 × 25 Å bundle consisting of six helices (PDB ID code 6lxt) (23). Its molecular weight is 40 kDa, close to the theoretical minimum size needed to achieve a reconstruction with near-atomic resolution by cryo-EM (27). To our knowledge, it has not yet been possible to determine structures of individual proteins <50 kDa to high resolution, with exception in the case of multimers (28, 29) or small RNA molecules (30). In addition, efforts extending the resolution limit of cryo-EM have largely focused on globular proteins (28, 29, 31, 32), perhaps because fibrous samples are more flexible, more susceptible to the issue of preferred orientation, and require thicker ice to bury the entire particle—all of which inevitably increase noise in the already extremely low-contrast and hard-to-align images. To overcome the size limit of single-particle cryo-EM, two strategies have been employed to increase the effective mass of the target protein, e.g. using antibodies/nanobodies/legobodies (33, 34) and molecular scaffolds (35–38). Since developing antibodies/nanobodies/legobodies can be time-consuming we resorted to the molecular scaffold approach. We first attempted to use existing scaffolds but were unable to engineer a linkage ensuring proper HR1HR2 bundle formation. We therefore designed a scaffold to efficiently determine structures of the postfusion HR1HR2 bundle and its mutants to near-atomic resolution by single-particle cryo-EM.Our high-resolution wild-type structure of the HR1HR2 bundle resolves uncertainties in some side-chain positions present in prior structures. Our HR1HR2 structures of SARS-CoV-2 variants reveal an overall architecture that is highly conserved, with only side-chain rearrangement for five point mutations and, for the Omicron variant containing three mutations in HR1, a slight shift of the HR2 backbone in a nonhelical region that interacts with HR1. These results suggest that interactions between HR1 and HR2 are excellent targets for disruption by broadly efficacious antiviral inhibitors. Moreover, our approach can be directly used to study the binding of potential HR2-based peptide inhibitors and adapted to study the postfusion bundles of other coronaviruses or other structurally similar viruses.     

Self-adaptive integration of photothermal and radiative cooling for continuous energy harvesting from the sun and outer space

The sun (∼6,000 K) and outer space (∼3 K) are two significant renewable thermodynamic resources for human beings on Earth. The solar thermal conversion by photothermal (PT) and harvesting the coldness of outer space by radiative cooling (RC) have already attracted tremendous interest. However, most of the PT and RC approaches are static and monofunctional, which can only provide heating or cooling respectively under sunlight or darkness. Herein, a spectrally self-adaptive absorber/emitter (SSA/E) with strong solar absorption and switchable emissivity within the atmospheric window (i.e., 8 to 13 μm) was developed for the dynamic combination of PT and RC, corresponding to continuously efficient energy harvesting from the sun and rejecting energy to the universe. The as-fabricated SSA/E not only can be heated to ∼170 °C above ambient temperature under sunshine but also be cooled to 20 °C below ambient temperature, and thermal modeling captures the high energy harvesting efficiency of the SSA/E, enabling new technological capabilities.

Heating and cooling are two kinds of significant end uses of thermal energy in society, which exist in various conditions (e.g., space/water heating, space cooling, and industrial processes) and account for 51% of the total final energy consumption (1). For example, the heating and cooling of buildings are responsible for nearly 48% of the building energy consumption, increasing to be the largest individual energy expense (2). Therefore, heat and cool harvesting relying on clean techniques from renewable energy resources has drawn remarkable attention from fields of engineering to material science because it has considerable potential for global energy conservation and greenhouse emission reduction. Thermodynamically, any heat transportation and work-generation process requires a temperature gradient. The hot sun (∼6,000 K) and cold outer space (∼3 K) are the ultimate heat source and heat sink for the Earth. Theoretical analysis reveals that maximal output work can be extracted from nonreciprocal systems based on the temperature difference between the sun and Earth (∼300 K) with an ultimate solar energy harvesting efficiency limit of 93.3%, while a maximal work of 153.1 W·m⁻² can also be obtained on the basis of temperature difference between the Earth and outer space (3, 4). Thus, the sun and outer space are two significant renewable thermodynamic resources for the Earth, which can be effectively utilized for clean heat and cool collection.Photothermal (PT) is a widely used solar thermal collection method that employs solar absorbers to capture solar photons and convert them to heat. Thermal analysis reveals that a good candidate for a solar absorber should have high solar absorptivity and low thermal emissivity simultaneously for efficient solar thermal collection. Various materials, including multilayer metal/ceramic films (5, 6), photonic crystals (7, 8), and metamaterials (9, 10), have been developed for spectrally selective solar absorbers and have been used for real-world applications. Meanwhile, radiative cooling (RC) has re-elicited considerable interest in recent years because it can passively provide clean cooling without any extra energy input (11–14). The waste heat of terrestrial objects can be continuously pumped into the cold outer space, relying on the transparent atmospheric window (i.e., 8 to 13 μm). So, high emissivity within the atmospheric window of materials is necessary for efficient RC, and excellent solar reflection is also important for RC under sunshine. Thus, different materials with the tailored spectrum, such as photonic structures (15–17), structure materials (18), energy-saving paints (19–21), and even metamaterials (22–24), have been reported for passive cooling. On the potential application level, RC implementations also span a range of fields, including passive cooling of buildings (25–27), thermal management of textiles and color surfaces (28–30), atmospheric water harvesting (31), and thermoelectric generation (32, 33). Although the reported PT and RC can generate heat and cold with high efficiency through different spectrally selective materials, most of the approaches are static and monofunctional, which can only provide heating or cooling under sunlight or darkness. Therefore, the dynamical integration of PT and RC for continuously efficient heat and cool harvesting is a new topic for the energy exploitation of the sun and outer space. The tunable combination of PT and RC hybrid utilization has been recently proposed, but mechanical methods such as switching (e.g., flip action) a PT absorber and an RC emitter manually (34) or changing the optical properties of the materials through extra force stimuli (35) are preferred.Herein, a smart strategy for the dynamic combination of daytime PT and nighttime RC is proposed, corresponding to continuously efficient energy harvesting from the sun and rejecting energy to the universe. A spectrally self-adaptive absorber/emitter (SSA/E) with solar absorption of over 0.8 and emissivity modulation capability of regulating from broadband emissivity of 0.25 within the mid-infrared (MIR) region to the selective high emissivity of 0.75 within the atmospheric window is designed and fabricated for the proof of the concept. Outdoor thermal experimental results demonstrate that the SSA/E can be heated to ∼170 °C above ambient temperature in the daytime PT mode and passively cooled to ∼20 °C below ambient temperature in the nighttime RC mode. Moreover, the heat and cool energy gains of the SSA/E system are respectively predicted to be 78% and 103% larger than those of the reference system that combines static and monofunctional PT absorber and RC emitter.     

The acquisition of cold sensitivity during TRPM8 ion channel evolution

To cope with temperature fluctuations, molecular thermosensors in animals play a pivotal role in accurately sensing ambient temperature. Transient receptor potential melastatin 8 (TRPM8) is the most established cold sensor. In order to understand how the evolutionary forces bestowed TRPM8 with cold sensitivity, insights into both emergence of cold sensing during evolution and the thermodynamic basis of cold activation are needed. Here, we show that the trpm8 gene evolved by forming and regulating two domains (MHR1-3 and pore domains), thus determining distinct cold-sensitive properties among vertebrate TRPM8 orthologs. The young trpm8 gene without function can be observed in the closest living relatives of tetrapods (lobe-finned fishes), while the mature MHR1-3 domain with independent cold sensitivity has formed in TRPM8s of amphibians and reptiles to enable channel activation by cold. Furthermore, positive selection in the TRPM8 pore domain that tuned the efficacy of cold activation appeared late among more advanced terrestrial tetrapods. Interestingly, the mature MHR1-3 domain is necessary for the regulatory mechanism of the pore domain in TRPM8 cold activation. Our results reveal the domain-based evolution for TRPM8 functions and suggest that the acquisition of cold sensitivity in TRPM8 facilitated terrestrial adaptation during the water-to-land transition.

Given that temperature influences all biological operations, the evolution of thermosensory adaptation is crucial in shaping the specialized temperature-dependent inhabitation of an organism. At the cellular level, thermosensory neurons in the dorsal root ganglia or trigeminal ganglia innervate the skin and transmit temperature information to the spinal cord and the brain. To bestow such neurons with thermal sensitivity, animals have a toolkit of temperature-sensitive ion channels located on the cell membrane at the molecular level. Accordingly, several members of the transient receptor potential (TRP) superfamily with steep thermosensitivity (referred to as thermoTRP) have attracted the general interest in the field of thermal biology, as they sufficiently cause steep changes in depolarizing currents upon either heating or cooling and thus are considered as the primary molecular sensors of temperature (1–4). Therefore, the evolutionary strategy for directly tuning the thermal activation in thermoTRPs can be employed by animals for their specialized thermosensory adaptation, as seen in vampire bats, pit-bearing snakes, platypus, penguins, squirrels, and camels (5–9).As heat sensation (warmth and extreme heat) provides the precondition of a fundamental and conserved biological survival process, the genes that encode heat sensors are considered ancient in many metazoan organisms. The annotation of trpv1 is consistently available in the genomes of fishes, insects, amphibians, reptiles, birds, and mammals. Despite the species-specific temperature-sensitive ranges, a growing number of studies have reported the functional convergence of these heat-sensitive thermoTRP orthologs at the protein level (10), suggesting the essential role of these channels in heat perception across species. Compared to heat sensors, the cold-sensitive thermoTRP likely evolved late. As the most established cold sensor responsive to low temperatures and cooling compounds, transient receptor potential melastatin 8 (TRPM8) was found in somatosensory neurons, and genetic ablation of trpm8 either in the neurons or mice led to a largely decreased cold sensitivity (4, 11–13). Interestingly, cold activation of amphibian TRPM8 has been tested (14), while sequencing efforts indicated the absence of the trpm8 gene in 12 fish species from 10 different orders (15). Several specific domains that may alter TRPM8 cold activation have been reported, including the pore domain, voltage sensing apparatus, and C terminus (8, 16–19). Notably, although the efficacy of cold activation is largely altered by residue substitutions in the pore domain, the channel mutants are still cold sensitive (8). Therefore, these findings based on domain/residue swapping among cold-sensitive TRPM8 orthologs may not draw an overall picture in functionally important domains responsible for cold sensitivity. How did the trpm8 gene originate? How did TRPM8 integrate and modulate cold sensitivity throughout evolution? The answers to such questions probably lead us to understand the evolution of temperature perception and identify the essential structural elements that shape TRPM8 cold activation.In this study, we show the presence of the young trpm8 gene in lobe-finned fishes, believed to be the ancestors that gave rise to all land vertebrates (20). Such a young type of trpm8 derived from the trpm2 exon shuffling was originated and formed during the expansion of lobe-finned fish genomes. By detecting the positive selection-rich domains, we described the formation of the thermosensitive MHR1-3 domain in amphibian and reptile species that enables TRPM8 to undergo conformational changes at low temperatures. Furthermore, we found that the TRPM8 pore domain of terrestrial vertebrates evolved to tune the efficacy of cold activation, in which a cold-sensitive MHR1-3 domain is indispensable to achieve such a modulatory mechanism. Together, our findings suggest that the trpm8 gene origination and formation of the TRPM8 MHR1-3 domain contributed to the transition of vertebrate life from water to land and that the efficacy of cold activation tuned by the TRPM8 pore domain diversified the setting of temperature-adaptive phenotypes in terrestrial vertebrates.     

Pan‐cancer analysis reveals sex‐specific signatures in the tumor microenvironment

The processes of cancer initiation, progression, and response to therapy are affected by the sex of cancer patients. Immunotherapy responses largely depend on the tumor microenvironment (TME), but how sex may shape some TME features, remains unknown. Here, we analyzed immune infiltration signatures across 19 cancer types from 1771 male and 1137 female patients in The Cancer Genome Atlas to evaluate how sex may affect the tumor mutational burden (TMB), immune scores, stromal scores, tumor purity, immune cells, immune checkpoint genes, and functional pathways in the TME. Pan‐cancer analyses showed higher TMB and tumor purity scores, as well as lower immune and stromal scores in male patients as compared to female patients. Lung adenocarcinoma, lung squamous carcinoma, kidney papillary carcinoma, and head and neck squamous carcinoma showed the most significant sex biases in terms of infiltrating immune cells, immune checkpoint gene expression, and functional pathways. We further focused on lung adenocarcinoma samples in order to identify and validate sex‐specific immune cell biomarkers with prognostic potential. Overall, sex may affect the tumor microenvironment, and sex‐specific TME biomarkers may help tailor cancer immunotherapy in certain cancer types.     

Highly covalent molecular cage based porous organic polymer: pore size control and pore property enhancement

It remains a great challenge to effectively control the pore size in porous organic polymers (POPs) because of the disordered linking modes. Herein, we used organic molecular cages (OMCs), possessing the properties of fixed intrinsic cavities, high numbers of reactive sites and dissolvable processability, as building blocks to construct a molecular cage-based POP (TPP-pOMC) with high valency through covalent cross coupling reaction. In the formed TPP-pOMC, the originating blocking pore channels of TPP-OMC were “turned on” and formed fixed pore channels (5.3 Å) corresponding to the connective intrinsic cavities of cages, and intermolecular pore channels (1.34 and 2.72 nm) between cages. Therefore, TPP-pOMC showed significant enhancement in Brunauer–Emmett–Teller (BET) surface area and CO₂ adsorption capacity.

By utilizing the cage to framework strategy, the blocking pores of the cage itself were “turned on” to construct a highly covalent molecular cage based porous organic polymer.     

色素上皮源性因子对缺血-再灌注视网膜神经节细胞的保护作用

目的:研究色素上皮源性因子(pigment epithelium derived factor,PEDF)对高眼压诱导的大鼠视网膜缺血-再灌注后视网膜神经节细胞的保护作用.方法:经眼角膜进行前房平衡盐水(BSS)灌注,维持眼内压110 mmHg,以阻止视网膜正常血液灌注.60 min后取出灌注针头,恢复视网膜正常血流,从而建立大鼠视网膜缺血-再灌注模型.实验分为正常非缺血组和视网膜缺血-再灌注组,后者又分为生理盐水注射对照组和PEDF注射实验组,再灌注模型建立后立即向实验组大鼠玻璃体腔内注射0.2 g/L PEDF 2 μL.实验对照组用同样方法注射等量生理盐水.分别于注射后2 d和7 d进行眼球摘除,对视网膜进行光学显微镜形态学观察和Fas原位杂交免疫学分析,探讨PEDF对缺血-再灌注视网膜神经节细胞的保护作用.结果:缺血-再灌注2 d时生理盐水注射组和PEDF注射组视网膜神经节细胞明显少于正常对照组(P＜0.01)和(P＜0.05),PEDF注射组视网膜神经节细胞数较生理盐水注射组明显较多,相比有显著性差异(P＜0.05);视网膜神经节细胞计数再灌注7 d后结果与2 d时类似.再灌注2 d生理盐水注射组Fas阳性染色细胞比PEDF注射组明显较多(P＜0.05),生理盐水组比PEDF注射组阳性细胞百分率明显较高(P＜O.01);再灌注7 d时两组Fas阳性细胞计数无明显差异.结论:视网膜缺血-再灌注后即刻行玻璃体腔内PEDF注射可以改善视网膜神经节细胞的损伤并有一定保护作用.     

VEGF siRNA对兔眼碱烧伤后角膜新生血管的抑制作用

目的:研究血管内皮生长因子小干扰RNA(VEGF small in-terfering RNA,VEGF siRNA)对兔眼碱烧伤后角膜新生血管的抑制作用。方法:普通家兔25只以碱烧伤法(1mol/LNaOH溶液)诱导角膜新生血管(CNV)生成。碱烧伤后立即以脂质体(LF2000)为载体,右眼球结膜下注射VEGF siRNA重组质粒,左眼球结膜下注射pSilencer 2.1-U6 hygro空白质粒作为阴性对照。碱烧伤后1,3,5,7,14d,形态学分析评价角膜新生血管的生长情况。结果:与对照眼相比,碱烧伤后球结膜下注射VEGF siRNA重组质粒,实验眼在各时间段(3,5,7,14d),CNV长度明显变短,面积明显变小,差异有统计学意义(P<0.05)。结论:VEGF siRNA能有效地抑制碱烧伤后CNV的形成。     

超声生物显微镜对慢性低眼压诊断和治疗的动态监测

目的:一组以UBM诊断为基础的慢性低眼压患者,经眼压、裂隙灯显微镜、眼底检查、UBM3~6mo的动态监测,了解低眼压的恢复情况,不同治疗方法的效果,说明UBM在低眼压诊断和治疗中的作用。方法:慢性低眼压45眼(眼压≤8mmHg),平均眼压为5.59(2~8)mmHg,病史超过1mo。所有患者均进行常规眼科检查,包括视力、裂隙灯、眼压检查、前房角镜、间接检眼镜、UBM检查,治疗分为药物组和手术组,于治疗后1wk;1,3和6mo进行随访检查。结果:经UBM检查有24眼(53%)为钝伤性低眼压,主要为睫状体脉络膜脱离;16眼(36%)为各种内眼手术后所致的低眼压;1例(0.04%)患者为风湿性心脏病继发双眼葡萄膜炎。基线眼压与治疗后随访眼压比较,均存在显著性差异(P<0.01)。末次眼压和基线眼压之间无相关性,而和病史及发病年龄均呈强的负相关性。42眼(93%)UBM检查有形态学结构改变,睫状体异常占87%。药物组和手术组的基线眼压分别为5.91和4.95mmHg,存在显著性差异(P<0.05),药物组病史明显长于手术组,而手术组发病年龄明显低于药物组,随访的末次眼压分别为10.42和11.25mmHg,无显著性差异。结论:经治疗两组眼压均有显著提高,手术组的患者病情明显重于药物组,治疗前眼压低,发病年龄小,但治疗后眼压效果相似。UBM无论在低眼压的诊断,还是治疗后监测中均有非常重要的作用。     

Simultaneous quantification of apolipoproteins A-I,E, and J in human plasma by LC-MS/MS for clinical application to diabetes mellitus complicated with cardiovascular disease

Apolipoproteins (Apos) play an important role in regulating plasma lipid concentration. Complex disorders of Apos are highly related with diabetes mellitus, cardiovascular and other diseases. Direct measures of lipoprotein fractions for risk assessment suffer from inaccuracy in the dyslipidemia and pathological states. Therefore, a reliable precise assay will be of high clinical utility. LC-MS/MS methods with multiple reaction monitoring modes have proven suitable for multiplexed quantification. We aimed to develop a simple, cost-effective and amenable LC-MS/MS assay for quantification of ApoA-I, ApoE and ApoJ in human plasma. Standards were constructed from substitute matrix and proteotypic peptides for external calibration and corresponding stable isotope labeled peptides were added as internal standards to remove matrix effects. Analytical validation of the assay included the assessment of linearity, accuracy (RE: −3.02% to 5.32%), intra-assay precision (RSD: 2.50% to 6.56%), inter-assay precision (RSD: 0.78% to 6.68%), spiking recovery rate (accuracy: 87.17% to 112.71%), matrix effect (accuracy: 88.03% to 114.87%), and reproducibility and repeatability of sample preparation (RSD: 1.95% to 7.26%). The performance of proteotypic peptides ApoA-I, ApoE and ApoJ was sufficient for triplex quantitation within a linear range from 16.26 to 1626.41 pmol mL⁻¹, 1.03 to 103.35 pmol mL⁻¹ and 0.86 to 86.46 pmol mL⁻¹ respectively. For all quantified peptides, the determination coefficient (R²) was >0.997. Besides, the validated LC-MS/MS method has been successfully applied to the quantification of plasma samples in diabetes mellitus and cardiovascular diseases. We anticipate that this assay may provide an alternative method for future clinical applications.

Simultaneous quantification of apolipoproteins A-I, E, and J in human plasma by LC-MS/MS.