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Proteome-wide multipoint internal calibration curves for mass spectrometry-based quantitative proteomics

ABSTRACT: Mass spectrometry (MS)-based proteomics is known for its high accuracy in quantifying peptides and proteins using various calibration strategies, including internal and external calibration curves. While external multi-point calibration curves are created from serial dilutions, they often fail to account for sample-specific matrix effects. In contrast, internal calibration curves account for sample matrix but face scalability and cost challenges for whole proteome analyses. In this manuscript we present a novel TMT-based multipoint internal calibration curve strategy, referred to as TMTCal, which enables the generation of internal calibration curves for all peptides identified within a proteome within a single experiment. We applied this strategy to human ovarian cancer cells to evaluate the linear quantitative responses of all the identified peptides and reveal the significant proteome changes associated with cisplatin treatment.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Ovary Cancer Cell Line

DISEASE(S): Malignant Neoplasm Of Ovary

SUBMITTER: Cristina Chiva

LAB HEAD: Eduard Sabidó

PROVIDER: PXD059628 | Pride | 2025-02-24

REPOSITORIES: pride

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			Action	DRS
	2020LF003-rep-1-ttest-isotopic-true.pdResult	Other
	2020LF003-rep-2-ttest-isotopic-true.pdResult	Other
	2020LF003-rep-3-ttest-isotopic-true.pdResult	Other
	2020LF003_CRIS_012_01_TMTCal_fraction1.raw	Raw
	2020LF003_CRIS_012_01_TMTCal_fraction11.raw	Raw

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Project description:Targeted proteomics has become the method of choice for biomarker validation in human biopsies due to its capacity to measure a set of peptides in multiple samples with high sensitivity, reproducibility, accuracy and precision. However, for targeted proteomics technologies to be transferred to clinical routine there is the need to reduce its complexity and make its procedures simpler, increase its throughput and improve its analytical performance. Biomarker peptide quantification with high accuracy and precision is one of the steps that still remains a challenge in clinical routine, mainly due to the difficulty to account for matrix effects (i.e. signal variation due to biological patient variability) and to establish a valid range of quantification for each analyte in each individual sample. Research-grade proteomics laboratories perform targeted peptide quantification with stable isotopically-labelled peptides (one per analyte), which are added to the samples and used as internal standards. This strategy, known as single-point calibration, enables to confidently assign the endogenous peptide and infer its quantity by direct comparison to the internal standard. This approach however assumes that both the endogenous and the internal standard peptides are within the linear range of quantification, which is not always granted as the linear dependency between peptide areas and concentrations only occurs in a limited sample-dependent range of concentrations. Although standard peptide abundances are adjusted to endogenous levels in low-throughput projects, this is not feasible when dealing with large cohorts of patients in clinical routine that exhibit a high variability of peptide abundances and different matrix effects. External multi-point standard curves in their different forms (calibrated, reverse) have been used to alleviate part of these limitations, but the requirement of a representative blank matrix and the fact that they do not account for matrix effects in individual samples nor establish a valid range of quantification for each individual sample limit their application. Here we present the Isotopologue Multiple-point Calibration (ImCal) quantification strategy, which uses a mix of isotopologue peptides to generate internal multiple-point calibration curves for each individual sample and to accurately quantify biomarker peptides in clinical applications without the need of expert supervision. ImCal relies on the use of five different isotopically-labelled peptides of different nominal mass―ranging from 10 to 39 Da mass shifts—mixed at different concentrations to be used as internal calibration curve for each endogenous peptide of interest

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Proteome-wide multipoint internal calibration curves for mass spectrometry-based quantitative proteomics

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