PXD030875

PXD030875 is an original dataset announced via ProteomeXchange.

Title	One-Pot Isotope Dilution PRM for Quantitative Protein Heterogeneity in Red Blood Cells
Description	Single cell and low-input cell proteomics to measure cellular heterogeneity remains experimental and requires further refinement for routine application. This study aimed to build on previous protocols to assess the utility of targeted proteomics for measuring heterogeneity in red blood cells (RBCs). Pioneering developments, nanodroplet processing in one-pot for trace samples (nanoPOTS) and automated preparation in one-pot for trace samples (autoPOTS), allow single cell proteomics on proteins with copy numbers <106. Here, the latter workflow was further simplified to eliminate robotic liquid handling instrument and modified (10-port) nano-LC switching valve, required for online solid phase extraction. When combined with isotope labeled synthetic peptide standards for targeted quantitative isotope dilution parallel reaction monitoring (PRM) LC-MS (IDMS-PRM), protein quantification was completed in 2.5 h from sample preparation to data analysis. To assess the utility of the protocol for measuring (protein) cellular heterogeneity, an IDMS-PRM assay was developed to measure endogenous N-terminal proteolytic peptide of hemoglobin beta subunit and it’s N-terminal valine carboxymethyl (CMV) adduct, which were quantified in single digit red blood cells (RBCs), isolated via limiting-dilution (LD). Substituting limiting dilution with a more sophisticated isolation strategy, achieved with the CellenONE single cell dispenser, the endogenous concentration of the N-terminal proteolytic peptide of hemoglobin beta subunit, and by inference the hemoglobin tetramer, was measured in single red blood cells (540-660 amol/RBC) which was comparable to the calculated SI reference range for mean corpuscular hemoglobin (390-540 amol/RBC). Moreover, it was observed repeated measures using low input cell numbers (5-25 RBC) could be used as an alternative and less rigorous strategy (compared to single RBC analysis) to measure protein heterogeneity. This heterogeneity was observed when %CMV was repeatedly measured using low input RBCs (5-25 RBCs) in specimens obtained from N = 10 individuals with clinically measured HbA1c values. In conclusion, a rapid and relatively simplified protocol has been developed for targeted quantification of proteins to measure red blood cell heterogeneity.
HostingRepository	PanoramaPublic
AnnounceDate	2024-02-06
AnnouncementXML	Submission_2024-02-06_15:53:29.180.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Supported dataset by repository
PrimarySubmitter	Azad Eshghi
SpeciesList	scientific name: Homo sapiens; NCBI TaxID: 9606;
ModificationList	Carboxymethyl; Oxidation; Label:13C(6)15N(2); Label:13C(6)15N(1)
Instrument	Orbitrap Fusion ETD; Orbitrap Exploris 480; 6495B Triple Quadrupole LC/MS

Revision	Datetime	Status	ChangeLog Entry
0	2022-01-10 21:44:02	ID requested
⏵ 1	2024-02-06 15:53:30	announced

Eshghi A, Xie X, Hardie D, Chen MX, Izaguirre F, Newman R, Zhu Y, Kelly RT, Goodlett DR, Sample Preparation Methods for Targeted Single-Cell Proteomics. J Proteome Res, 22(6):1589-1602(2023) [pubmed]

submitter keyword: Single cell proteomics, quantitative, One-pot, hemoglobin, erythrocyte, red blood cell, HbA1c, glycated hemoglobin, carboxymethyl hemoglobin.

David R. Goodlett
contact affiliation	University of Victoria
contact email	goodlett@uvic.ca
lab head
Azad Eshghi
contact affiliation	University of Victoria, Victoria, BC, Canada
contact email	azad@proteincentre.com
dataset submitter

Panorama Public dataset URI