title stringlengths 1 1.19k | keywords stringlengths 0 668 | concept stringlengths 0 909 | paragraph stringlengths 0 61.8k | PMID stringlengths 10 11 |
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DBS and venous characteristics associated with agreement between measurements | SAID | Of the DBS characteristics covariates tested, all impacted the association between serum and DBS measurements of at least one analyte. This means that models with an interaction between DBS values and the covariate in question significantly differed to the same nested model without said interaction. The number of spots... | PMC10415328 | |
Using DBS data to assess disease prevalence | overestimation of disease, diabetes | DISEASE, DIABETES | Using total cholesterol, HbA1c and CRP data, we compared the prevalence of disease in venous and venous-equivalent DBS. Comparable measurements should lead to a similar prevalence of disease in nurse and self-collection mode. However, participants who reported doctor diagnosed diabetes were less likely to provide a ven... | PMC10415328 |
Discussion | hypercholesterolaemia | DISEASE, HYPERCHOLESTEROLAEMIA | Our findings suggest that, while response rates for self-collected DBS samples are around half that in a nurse visit, characteristics of participants are unbiased with respect to educational attainment, self-rated health and longstanding illness. While measurement quality of HbA1c and HDL-cholesterol may be inconsisten... | PMC10415328 |
Supplementary Information | The online version contains supplementary material available at 10.1038/s41598-023-39674-6. | PMC10415328 | ||
Acknowledgements | We would like to thank Shavanthi Rajatileka and Melissa Smart for their significant contribution to the design and management of IP12, and Annette Jäckle for her contribution as Director of the Innovation Panel. | PMC10415328 | ||
Author contributions | M.K. conceived the idea for this paper and led the design with input from A.A., T.A.B., M.B., J.B. and T.C. K.S.J., A.K. and D.A.P. designed and led the analyte measurements. A.A. undertook all data analysis with input from M.K. M.K. and A.A. drafted the paper, with all authors critically revising the manuscript. All a... | PMC10415328 | ||
Funding | MK, AA, TAB, JB, TFC, MB and data collection for this study is funded by ESRC (ES/N00812X/1). The funders had no role in the study design, data collection, data analysis, data interpretation, or writing of the report. Understanding Society:UKHLS is an initiative funded by the Economic and Social Research Council (ES/S0... | PMC10415328 | ||
Data availability | Data from the | PMC10415328 | ||
Competing interests | The authors declare no competing interests. | PMC10415328 | ||
References | PMC10415328 | |||
BACKGROUND: | premature cardiovascular events | HOMOZYGOUS FAMILIAL HYPERCHOLESTEROLEMIA, HOMOZYGOUS FAMILIAL HYPERCHOLESTEROLEMIA, GENETIC DISEASE, HOMOZYGOUS FAMILIAL HYPERCHOLESTEROLEMIA | Homozygous familial hypercholesterolemia is a genetic disease characterized by extremely high levels of low-density lipoprotein cholesterol (LDL-C) and a high risk of premature cardiovascular events. The proof-of-concept study ORION-2 (A Study of Inclisiran in Participants With Homozygous Familial Hypercholesterolemia)... | PMC10815002 |
METHODS: | ORION-5 was a phase 3, 2-part, multicenter study in 56 patients with homozygous familial hypercholesterolemia and elevated LDL-C levels despite maximum tolerated doses of LDL-C–lowering therapies with or without lipoprotein apheresis. Patients eligible for part 1 (double-blind, 6 months) were randomized 2:1 to receive ... | PMC10815002 | ||
RESULTS: | The mean age of the patients was 42.7 years, and 60.7% were women. The mean baseline LDL-C levels were 294.0 mg/dL and 356.7 mg/dL in the inclisiran and placebo groups, respectively. The placebo-corrected percentage change in LDL-C level from baseline to day 150 was −1.68% (95% CI, −29.19% to 25.83%; | PMC10815002 | ||
CONCLUSIONS: | HOMOZYGOUS FAMILIAL HYPERCHOLESTEROLEMIA | Inclisiran treatment did not reduce LDL-C levels in patients with homozygous familial hypercholesterolemia despite substantial lowering of PCSK9 levels. Inclisiran was well-tolerated, and the safety findings were consistent with previously reported studies and the overall safety profile. | PMC10815002 | |
REGISTRATION: | URL: | PMC10815002 | ||
Clinical Perspective | PMC10815002 | |||
What Is New? | This 24-month study evaluated the efficacy, safety, and tolerability of inclisiran in patients with homozygous familial hypercholesterolemia and elevated low-density lipoprotein cholesterol (LDL-C) levels despite maximum tolerated doses of LDL-C–lowering therapies with or without lipoprotein apheresis.There was no stat... | PMC10815002 | ||
What Are the Clinical Implications? | LDL-C reduction | GENETIC DISEASE, HOMOZYGOUS FAMILIAL HYPERCHOLESTEROLEMIA, HOFH, HOMOZYGOUS FAMILIAL HYPERCHOLESTEROLEMIA | Although this study did not show a statistically significant reduction in LDL-C level in patients with homozygous familial hypercholesterolemia, inclisiran demonstrated a sustained reduction in PCSK9 levels and was well-tolerated, with a safety profile consistent with previous studies.Despite the lack of significant LD... | PMC10815002 |
METHODS | The data that support the findings of this study are available from the corresponding author upon reasonable request. | PMC10815002 | ||
Study Design and Treatment | ORION-5 (URL: The study had 2 sequential parts. Part 1 consisted of a 6-month double-blind placebo-controlled phase in which eligible patients were randomized 2:1 to receive either 300 mg of inclisiran sodium (equivalent to 284 mg of inclisiran) subcutaneously or placebo at day 1 and day 90. Part 2 consisted of an 18-m... | PMC10815002 | ||
Study Population | xanthoma | XANTHOMA, HETEROZYGOUS FAMILIAL HYPERCHOLESTEROLEMIA, HOFH | The study included participants aged ≥18 years with genetic confirmation or clinical diagnosis of HoFH on the basis of a history of an untreated LDL-C concentration >500 mg/dL (13 mmol/L) together with either xanthoma before 10 years of age or evidence of heterozygous familial hypercholesterolemia in both parents. Pati... | PMC10815002 |
Genotyping | Genotyping was performed by a central laboratory or was available from the medical records of the study population. On the basis of this assessment, patients were categorized as homozygous | PMC10815002 | ||
Ethical Considerations | ORION-5 was conducted in accordance with the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use E6 guideline for Good Clinical Practice, which has its origin from the Declaration of Helsinki. The study protocol and all amendments were approved by the independent ethics c... | PMC10815002 | ||
Data Sharing Statement | Novartis is committed to sharing access to patient-level data and supporting clinical documents from eligible studies with qualified external researchers. These requests are reviewed and approved by an independent review panel on the basis of scientific merit. All data provided are anonymized to respect the privacy of ... | PMC10815002 | ||
Efficacy End Points | SECONDARY, HOFH | The primary end point was the percentage change in LDL-C level from baseline to day 150 (part 1 of the study). Key secondary end points were the absolute change in LDL-C level from baseline to day 150 and the percentage change in apoB (apolipoprotein B100), non–high-density lipoprotein cholesterol (non-HDL-C), and tota... | PMC10815002 | |
Safety End Points | ADVERSE EVENTS | The safety and tolerability profile of inclisiran was measured by adverse events (AEs), serious AEs, and clinical laboratory values. In addition, an anti-inclisiran antibody analysis was performed. AEs were assessed from baseline to each assessment time up to day 720. Data captured during part 1 (up to day 180) were pr... | PMC10815002 | |
Statistical Analysis | PMC10815002 | |||
Sample Size Calculation | It was calculated that a sample size of at least 45 patients (randomized 2:1 to inclisiran:placebo), with at least 30 patients in the inclisiran arm, would provide >80% power to detect a 20% reduction (assuming an SD of 20% in each arm) in placebo-corrected LDL-C levels from baseline to day 150 in the inclisiran group ... | PMC10815002 | ||
Missing Data Imputation | SECONDARY | Missing values in part 1 were imputed for the primary and key secondary end points using a prespecified multiple imputation (a total of 100 imputed data sets) washout model. The washout model can be thought of as a modified control-based pattern–mixture model. This was used to explore the possibility of data missing no... | PMC10815002 | |
Efficacy Analysis | SECONDARY | The primary analysis was conducted on the intent-to-treat population and based on an ANCOVA model on the percentage change in LDL-C level from baseline to day 150 on each multiply imputed data set (100 total). The model included the part 1 treatment group as a fixed effect and the baseline LDL-C level as a covariate. T... | PMC10815002 | |
Safety Analysis | Safety variables were summarized descriptively by parts and treatment groups. Safety was analyzed in the safety population comprising all patients who received ≥1 dose of the study drug using the standard Medical Dictionary for Regulatory Activities nomenclature.All analyses were performed with SAS software (version 9.... | PMC10815002 | ||
RESULTS | PMC10815002 | |||
Baseline Demographic Characteristics | death, ASCVD | ASCVD | Overall, 56 patients were enrolled, of whom 37 were randomized to the inclisiran group and 19 to the placebo group. A total of 53 patients (94.6%) completed part 1, 47 of whom (88.7%) also completed part 2. The reasons for discontinuation were death, withdrawal of consent, or “other” (Overall, the mean (SD) age of pati... | PMC10815002 |
Key Secondary End Points | The placebo-corrected absolute change in LDL-C level from baseline to day 150 was 6.47 mg/dL, which was not statistically significant ( | PMC10815002 | ||
Other Secondary End Points | The placebo-corrected absolute and percentage changes from baseline by visit for LDL-C, apoB, non–HDL-C, lipoprotein(a), and total cholesterol levels were not statistically significant at any time point.The reductions in PCSK9 levels from baseline to days 90, 150, and 180 were consistently greater in the inclisiran gro... | PMC10815002 | ||
Safety Evaluation | pyrexia, deaths, erythema, diarrhea | VIRAL RESPIRATORY TRACT INFECTION, ADVERSE EVENT, SUDDEN CARDIAC DEATH, CORONAVIRUS INFECTION, ERYTHEMA, VIRAL PNEUMONIA, MULTIPLE ORGAN DYSFUNCTION SYNDROME, EVENTS | A summary of the treatment-emergent AEs and the treatment-emergent serious AEs in part 1 and in part 2 is shown in Table Treatment-Emergent Adverse Events (Safety Population)The most commonly reported treatment-emergent AEs did not differ between treatment groups in either part of the study. In part 1, the most common ... | PMC10815002 |
DISCUSSION | SECONDARY, HOFH | ORION-5 did not meet its primary objective of detecting a statistically significant difference in the percentage change in LDL-C levels from baseline to day 150 between the inclisiran and placebo groups. Placebo-corrected changes in PCSK9 levels from baseline to day 150 were observed with inclisiran treatment and were ... | PMC10815002 | |
Conclusions | HOFH | In this study, although inclisiran treatment did not result in a statistically significant reduction in LDL-C level compared with placebo, the sustained reduction in PCSK9 levels demonstrates target engagement with inclisiran even in patients with HoFH. Inclisiran was well-tolerated, and the safety findings are consist... | PMC10815002 | |
ARTICLE INFORMATION | PMC10815002 | |||
Acknowledgments | The authors thank Ganesh Pedgaonkar, PhD, Vennila Dharman, MBBS, and Ritika Paul, MSc, MPhil, of Novartis Healthcare Pvt Ltd, for providing medical writing support in accordance with Good Publication Practice 2022 guidelines (Good Publication Practice Guidelines for Company-Sponsored Biomedical Research; 2022 Update; | PMC10815002 | ||
Sources of Funding | Novartis Pharma AG initiated and provided funding for this study and provided medical writing and editorial support in the development of the manuscript. The funder was also involved in study design and data collection, analysis, and interpretation, as well as review and feedback on the manuscript. The authors, who inc... | PMC10815002 | ||
Disclosures | Dr Raal received consulting fees and honoraria from Amgen, Novartis Pharmaceuticals, Regeneron Pharmaceuticals, and LIB Therapeutics. Dr Durst received consulting fees and honoraria from Novartis and Sanofi, lecturing fees from Novartis, and honoraria from Medison. Drs Bi, Talloczy, Maheux, and Lesogor are full-time em... | PMC10815002 | ||
Supplemental Material | AppendixTable S1Figures S1–S3 | PMC10815002 | ||
Supplementary Material | PMC10815002 | |||
REFERENCES | PMC10815002 | |||
Methods | An external rehearsal pilot cluster randomised controlled trial in four NHS Trusts (clusters) in England, UK. Clusters were randomised to intervention (where all eligible midwives received the intervention) or control (no intervention delivered) arms. A random sample of 100 midwives were invited to complete questionnai... | PMC9858407 | ||
Results | Seventy-four midwives consented and 68 returned questionnaires. Pre-intervention, self-efficacy for support/intervention-related behaviours scored lowest. In controls, there was limited difference between the pre- and post-intervention scores. Post-intervention, mean (SD) scores were consistently higher among intervent... | PMC9858407 | ||
Conclusions | This study supports the theoretical models used to develop GLOWING, where low self-efficacy was a core implementation barrier. Results suggest that GLOWING successfully targets self-efficacy, potentially with a positive impact on guideline implementation. A definitive trial is required to determine effectiveness. | PMC9858407 | ||
Data Availability | Data cannot be shared publicly because ethical approval and consent did not specify this and some participants could potentially be identifiable from the data. Requests to access the data must be made to the approving Ethics Committee (Proportionate Review Sub-committee of the Yorkshire & The Humber—South Yorkshire Res... | PMC9858407 | ||
Introduction | Obesity, weight gain, weight retention | OBESITY | More than half of women enter pregnancy with a body mass index (BMI) in the overweight (BMI≥25kg/mWeight management interventions delivered during pregnancy can significantly improve women’s diet and physical activity (PA) behaviours, reduce GWG and postnatal weight retention, and reduce the risk of developing some adv... | PMC9858407 |
Design, setting and participants | The details of the GLOWING intervention methods have been described elsewhere [ | PMC9858407 | ||
CONSORT 2010 flow diagram for cluster trials. | * Intervention arm: n = 33 midwives returned ≥1 questionnaire: n = 32 pre-intervention, n = 15 post-intervention, n = 14 both ** Control arm: n = 35 midwives returned ≥1 questionnaire: n = 34 pre-intervention, n = 34 post-intervention, n = 33 both. | PMC9858407 | ||
Intervention development and delivery | We followed a four-step approach for developing theory-informed implementation interventions [ | PMC9858407 | ||
SCT theoretical model for implementation of guideline recommendations for weight management in pregnancy: Communication-related behaviours. | SCT = social cognitive theory. Self-efficacy, outcome expectancies, goals/intentions and environment are SCT constructs relating to behaviours. Knowledge was identified as an important additional construct in the evidence base having an influence on self-efficacy. Bold and larger font size represent the key barriers to... | PMC9858407 | ||
SCT theoretical model for implementation of guideline recommendations for weight management in pregnancy: Support/intervention-related behaviours. | SCT = social cognitive theory. Self-efficacy, outcome expectancies, goals/intentions and environment are SCT constructs relating to behaviours. Knowledge and attitudes were identified as an important additional construct in the evidence base having an influence on self-efficacy and outcome expectancies. Bold and larger... | PMC9858407 | ||
Data collection | obesity | OBESITY | Data reported in this paper are from the questionnaires that midwives completed pre- and post-intervention. There were no existing validated questionnaires that could be used to measure the SCT constructs as the questions had to be tailored to the guideline specific behaviours. The NICE guideline recommendations releva... | PMC9858407 |
Data analysis | obesity | OBESITY | All data entry was carried out in duplicate. Two people entered the data independently, compared the data entered to identify potential data entry errors, and any discrepancies in data entry were validated against the original questionnaires. Descriptive analysis was carried out to compare the data with the theoretical... | PMC9858407 |
SCT questionnaires | The data for the behaviour items and SCT constructs were transformed to a 0–100 scale to enable comparisons across all constructs. The sum scores for the 7-point Likert scales were calculated by combining the individual questions within the two behaviour categories and for each SCT construct (The same process was carri... | PMC9858407 | ||
Research ethics | The study was approved by the Proportionate Review Sub-committee of the Yorkshire & The Humber—South Yorkshire Research Ethics Committee (ref: 15/YH/0565, 16/12/2015). All midwives were provided with an information sheet explaining the study, and written informed consent was provided before completing the questionnaire... | PMC9858407 | ||
Results | PMC9858407 | |||
Participants | overweight, SD | One hundred midwives were randomly selected to complete the GLOWING questionnaires (n = 49 intervention, n = 51 control arm), 74 consented and 68 (92%) of those who consented returned questionnaires: 33 (87%) midwives in the intervention and 35 (97%) in the control arm. Of these, 47 (69%) returned both the pre- and pos... | PMC9858407 | |
Personal and professional characteristics of midwives completing pre-intervention questionnaires. | obesity | OBESITY | SD = standard deviationWhen asked to consider whether their own weight made it easier, harder, or made no difference to having weight-related communication with pregnant women, the greatest proportion of midwives felt that their weight made it easier to discuss women’s weight status (43.1%) and obesity risks (50.0%) ( | PMC9858407 |
Questionnaire data | The Cronbach’s Alpha analysis demonstrated good internal consistency for communication-related behaviours and support/intervention-related behaviours for all SCT constructs (Cronbach’s Alpha ranging from 0.82–0.96; | PMC9858407 | ||
Comparing pre-intervention data with theoretical models | Pre-intervention, the midwives’ scores for the communication-related behaviours were generally higher overall than the scores for the support/intervention-related behaviours ( | PMC9858407 | ||
Comparing intervention and control arms | The descriptive statistics for pre- and post-intervention data are shown in | PMC9858407 | ||
Discussion | obesity, gestational diabetes | OBESITY, GESTATIONAL DIABETES | This paper reports the descriptive results of the GLOWING pilot trial relating to midwives reporting of their self-efficacy, outcome expectancies, intentions and routine behaviours in the context of UK guidelines for weight management during pregnancy. The data reported in this study provide some proof of concept for t... | PMC9858407 |
Conclusions | The GLOWING pilot trial data provides proof of concept of the theoretical models used to inform its development. The descriptive data reported in this paper suggests that the intervention may be successfully targeting self-efficacy as it was designed to do. A definitive trial with adequate power is required to determin... | PMC9858407 | ||
Supporting information | (DOCX)Click here for additional data file. | PMC9858407 | ||
Flowchart of NICE Guideline behaviours relevant to midwifery practice grouped into thematic behaviour categories and sub-categories for communication-related behaviours and behaviour support/intervention-related behaviours. | Note, Behaviour categories and sub-categories are: 1. Communication-related behaviours (sub-categories: weight communication and risk communication) 2. Support and intervention-related behaviours (sub-categories diet and nutrition, physical activity, weight management, referrals and signposting).(DOCX)Click here for ad... | PMC9858407 | ||
Standards for reporting implementation studies: The StaRI checklist. | (DOC)Click here for additional data file. | PMC9858407 | ||
Adapted NICE guideline recommended behaviours developed for the GLOWING questionnaire. | (DOCX)Click here for additional data file. | PMC9858407 | ||
Sum score behaviour categories derived from the questionnaire items for each SCT construct. | * Questions required reverse coding.(DOCX)Click here for additional data file. | PMC9858407 | ||
Midwife characteristics and scores for the behaviour categories and Social Cognitive Theory (SCT) constructs, comparing those who returned one or both questionnaires. | *Note, 21 midwives only returned one questionnaire, two of these only returned their post-intervention questionnaire therefore have missing pre-intervention data.(DOCX)Click here for additional data file. | PMC9858407 | ||
Midwives perceptions of the impact of their own weight on the level of difficulty the experience discussing weight status and risks of obesity with pregnant women. | HW = healthy weight, OW = slightly overweight/very overweight *HW in the intervention arm includes one midwife who perceived their own weight as slightly underweight.(DOCX)Click here for additional data file. | PMC9858407 | ||
Internal validity of the questionnaire items for each behaviour category and social cognitive theory construct. | *Communication-related behaviours include weight communication and risk communication; support/intervention-related behaviours include diet and nutrition, physical activity, weight management, and referrals and signposting.(DOCX)Click here for additional data file. | PMC9858407 | ||
A comparison of pre- and post-intervention scores for communication- and support/intervention-related behaviour sub-categories and social cognitive theory constructs. | SD = standard deviation * Due to the limited number of diet and nutrition questions for self-efficacy and outcome expectancies, these were combined with the physical activity questions when creating the sum scores for these constructs.(DOCX)Click here for additional data file.We would like to thank Anita Tibbs, Phoebe ... | PMC9858407 | ||
Abstract | PMC9825269 | |||
STUDY QUESTION | Does an estradiol-based combined oral contraceptive (COC) have a milder effect on the serum proteome than an ethinylestradiol (EE)-based COC or dienogest (DNG) only? | PMC9825269 | ||
SUMMARY ANSWER | The changes in serum proteome were multifold after the use of a synthetic EE-based COC compared to natural estrogen COC or progestin-only preparation. | PMC9825269 | ||
WHAT IS KNOWN ALREADY | blood coagulation | INFLAMMATION | EE-based COCs widely affect metabolism, inflammation, hepatic protein synthesis and blood coagulation. Studies comparing serum proteomes after the use of COCs containing EE and natural estrogens are lacking. | PMC9825269 |
STUDY DESIGN, SIZE, DURATION | This was a spin-off from a randomized, controlled, two-center clinical trial. Women (n = 59) were randomized to use either EE + DNG, estradiol valerate (EV) + DNG or DNG only continuously for 9 weeks. | PMC9825269 | ||
PARTICIPANTS/MATERIALS, SETTING, METHODS | Participants were healthy, young, white volunteer women. Serum samples were collected before and after 9 weeks of hormonal exposure. Samples from 44 women were available for analysis (EE + DNG n = 14, EV + DNG n = 16 and DNG only n = 14). Serum proteins were analyzed by quantitative, discovery-type label-free proteomic... | PMC9825269 | ||
MAIN RESULTS AND THE ROLE OF CHANCE | ACUTE PHASE RESPONSE | Altogether, 446 proteins/protein families with two or more unique peptides were detected and quantified. The number of proteins/families that altered over the 9-week period within the study groups was 121 for EE + DNG and 5 for EV + DNG, while no changes were detected for DNG only. When alterations were compared betwee... | PMC9825269 | |
LARGE SCALE DATA | Data are available via ProteomeXchange with identifiers PXD033617 (low abundance fraction) and PXD033618 (high abundance fraction). | PMC9825269 | ||
LIMITATIONS, REASONS FOR CAUTION | The power analysis of the trial was not based on the proteomic analysis of this spin-off study. In the future, targeted proteomic analysis with samples from another trial should be carried out in order to confirm the results. | PMC9825269 | ||
WIDER IMPLICATIONS OF THE FINDINGS | The EE-based COC exerted a broader effect on the serum proteome than the EV-based COC or the DNG-only preparation. These results demonstrate that the effects of EE in COCs go far beyond the established endpoint markers of estrogen action, while the EV combination is closer to the progestin-only preparation. The study i... | PMC9825269 | ||
STUDY FUNDING/COMPETING INTEREST(S) | EVENTS | Funding for this researcher-initiated study was obtained from the Helsinki University Hospital research funds, the Hospital District of Helsinki and Uusimaa, the Sigrid Juselius Foundation, the Academy of Finland, the Finnish Medical Association, the University of Oulu Graduate School, the Emil Aaltonen Foundation, the... | PMC9825269 | |
TRIAL REGISTRATION NUMBER | ClinicalTrials.gov NCT02352090 | PMC9825269 | ||
TRIAL REGISTRATION DATE | 27 January 2015 | PMC9825269 | ||
DATE OF FIRST PATIENT’S ENROLMENT | 1 April 2015 | PMC9825269 | ||
Introduction | ADVERSE EVENTS | In addition to the contraceptive effect, combined oral contraceptives (COCs) have vast effects on female physiology. In the liver, COCs stimulate the synthesis of steroid-binding globulins, such as sex hormone-binding globulin (SHBG), thereby affecting circulating, free steroid levels (The net effect of COCs results fr... | PMC9825269 | |
Materials and methods | This randomized, controlled, investigator-initiated clinical trial was conducted at Helsinki and Oulu University Hospitals, Finland, between April 2015 and January 2018. The study protocol has been described previously ( | PMC9825269 | ||
Subjects | Seventy-seven healthy women volunteered for the study (
| PMC9825269 | ||
Intervention | Participants were randomized to use either EE + DNG (Valette | PMC9825269 | ||
Serum proteomic analysis | Serum samples were stored at −70°C until analysis. High-abundance proteins and their protein complexes were affinity-purified from the samples, and the samples were divided into two pools: high- and low-abundance proteins. Human serum albumin was further removed from the high-abundance samples. Proteins were digested w... | PMC9825269 | ||
Affinity chromatography | An Agilent Technologies (Santa Clara, CA, USA) Multiple Affinity Removal Column Human 14 (Cat No 5188-6557) was used in the affinity chromatography, according to the manufacturer’s instructions. Samples were diluted four-fold with buffer A. Fourteen proteins and their complexes (i.e. ‘high-abundance proteins’) were ret... | PMC9825269 | ||
Desalting and albumin removal from high-abundance samples | All samples were desalted using Thermo Fisher Scientific (Waltham, MA, USA) Zeba Spin desalting 96-well plates (Cat No 89808) to change the buffer to 25 mM Trizma BASE + 75 mM NaCl pH 7–8. The high-abundance samples were albumin-depleted with a Thermo Fisher Scientific Pierce Albumin Depletion Kit (Cat No 85160). Prote... | PMC9825269 | ||
Trypsin digestion | Proteins in the samples were trypsin-digested with the aid of Waters Corporation (Milford, MA, USA) 0.2% surfactant RapiGest™ SF. Samples were boiled for 10 min, and proteins were reduced by adding 0.5 M 1,4-dithiothreitol to a final concentration of 5 mM, vortexed and incubated for 30 min in 60°C water bath. In the ne... | PMC9825269 | ||
Mass spectrometry | Five hundred nanograms of the Hi3 spiked peptide mixture was injected to the Waters Corporation Synapt G2-Si HDMS and nanoACQUITY system. The system was equipped with Trap Column Symmetry C18, nanoACQUITY 10K 2G V/M Trap Column, 100 Å, 5 µm, 180 µm × 20 mm (Cat No 186006527) and analytical ultra-performance liquid chro... | PMC9825269 | ||
Peptide identification and quantitation | The raw data were imported to Progenesis QI for proteomics (Nonlinear Dynamics, La Jolla, CA, USA), and simultaneously mass corrected with the leucine–enkephalin function. Proteinlynx Global Server (Waters Corporation) was used to identify peptides from the UDMS | PMC9825269 |
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