Dataset Information

3D-Printed Device for Consistent and High-Throughput Peptide Sample Clean-up Using StageTips

ABSTRACT: Mass spectrometry (MS) is a fundamental technology for the study of proteomics. A core technique for MS analysis is solid-phase extraction (SPE) for the removal of contaminants and sample concentration, and StageTips are one of the most commonly used devices used for SPE in modern proteomics workflows. Here, we detail a device that can accommodate up to 96 StageTips enabling high-speed centrifugal-based processing of StageTips, including collection into PCR tubes, or 96-well plates, simplifying the processing of samples for MS analysis. The device can be rapidly and economically manufactured using a 3D printer employing fused deposition modelling. We show that high impact polystyrene (HIPS) is a particularly suitable material for building this device, providing good chemical resistance and is able to withstand high centrifugal speeds to enable repeated use. We provide detailed schematics as well as the corresponding CAD files for the community, enabling reproduction of the device on any common 3D printer model. Using an optimized protocol, we show that our tip spinner device provides similar peptide identifications and comparable inter-sample consistency than previously established centrifugation-based cleanup on individual StageTips. As such, this apparatus facilitates greater throughput in sample preparation for mass spectrometry, without detracting from sample quality.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Mark Larance

LAB HEAD: Mark Larance

PROVIDER: PXD009324 | Pride | 2019-03-21

REPOSITORIES: Pride

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Publications

Proteomic Analysis of Human Plasma during Intermittent Fasting.

Harney Dylan J DJ Hutchison Amy T AT Hatchwell Luke L Humphrey Sean J SJ James David E DE Hocking Samantha S Heilbronn Leonie K LK Larance Mark M

Journal of proteome research 20190401 5

Intermittent fasting (IF) increases lifespan and decreases metabolic disease phenotypes and cancer risk in model organisms, but the health benefits of IF in humans are less clear. Human plasma derived from clinical trials is one of the most difficult sample sets to analyze using mass spectrometry-based proteomics due to the extensive sample preparation required and the need to process many samples to achieve statistical significance. Here, we describe an optimized and accessible device (Spin96) ...[more]

PMID: 30892045

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Project description:Background: Mortality from preeclampsia (PE) and PE-associated morbidities are 3-to 5-fold higher in persons of African ancestry than in those of Asian and European ancestries. The placenta is central to the etiology of PE. However, how and to what extent the placenta contributes to worse PE outcomes in persons of African ancestry is yet to be fully elucidated. Objective: We aimed to identify molecular pathways that are unique or enriched in placentas of parturient persons of African ancestry with PE with severe features (sPE) compared to those of Asian and European ancestry with sPE. Study design: Bulk RNA sequencing was performed on 50 placentas from parturient persons with sPE of African (n=9), Asian (n=18) and European (n=23) ancestries and 73 normotensive controls of African (n=9), Asian (n=15) and European (n=49) ancestries. Results: Metabolism, hormone regulation and hypoxia/angiogenesis genes, previously described to be upregulated in PE, including: LEP, PAPPA2, INHA, FSTL3, FLT1, PHYHIP and ENG, were upregulated in sPE across ancestries, with high expression of FSTL3 being additionally associated with intrauterine growth restriction (p<.0001). Notably, LEP, FLT1 and PHYHIP were more highly upregulated in sPE placentas from parturient persons of African versus Asian ancestry. Genes associated with allograft rejection and adaptive immune response were selectively upregulated in placentas from African ancestry parturitions and not in those of Asian and European ancestries. Among the allograft rejection/adaptive immune response genes, IL3RA was of particular interest because the patient with the highest placental IL3RA level, a woman of African ancestry with IL3RA levels 4.5-fold above the average for African ancestry parturitions with sPE, developed postpartum cardiomyopathy, and was the only patient out of 123, that developed this condition. Interestingly, the sPE patients of Asian and European ancestries with the highest IL3RA levels for their ancestries developed unexplained tachycardia peripartum, necessitating echocardiography in the European ancestry patient. The association between elevated placental IL3RA levels and unexplained tachycardia or peripartum cardiomyopathy was found to be significant in the 50 sPE patients (p=.0005). Conclusions: African ancestry parturitions are associated with higher placental upregulation of metabolism (LEP) and hypoxia/angiogenesis (FLT1) genes, and selective upregulation of allograft rejection/adaptive immune response genes, including IL3RA. High placental expression of IL3RA may predict worse maternal cardiovascular outcomes, including peripartum cardiomyopathy. Studies evaluating placental IL3RA levels in peripartum cardiomyopathy cohorts are therefore warranted, as are broader studies evaluating placental factors in maternal cardiovascular outcomes postpartum.

Dataset Information

3D-Printed Device for Consistent and High-Throughput Peptide Sample Clean-up Using StageTips

Publications

Proteomic Analysis of Human Plasma during Intermittent Fasting.

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