Abstract
Analysis by liquid chromatography and tandem mass spectrometry (LC-MS/MS) can identify and quantify thousands of proteins in microgram-level samples, such as those comprised of thousands of cells. This process, however, remains challenging for smaller samples, such as the proteomes of single mammalian cells, because reduced protein levels reduce the number of confidently sequenced peptides. To alleviate this reduction, we developed Data-driven Alignment of Retention Times for IDentification (DART-ID). This method implements global retention time (RT) alignment to infer peptide RTs across experiments. DART-ID then incor-porates the global RT-estimates within a principled Bayesian framework to increase the confidence in correct peptide-spectrum-matches and decrease confidence in incorrect peptide-spectrum-matches. Applying DART-ID to hundreds of monocyte and T-cell samples pre-pared by the Single Cell Proteomics by Mass Spectrometry (SCoPE-MS) design increased the number of data points by 30 − 50% at 1% FDR, and thus decreased missing data. Quantification benchmarks indicate excellent quantification of peptides upgraded by DART-ID and support their utility for downstream analysis, such as identifying cell types and cell-type specific proteins. The additional datapoints provided by DART-ID boost the statistical power and double the number of proteins identified as differentially abundant in monocytes and T-cells. DART-ID can be applied to diverse experimental designs and is freely available at http://github.com/SlavovLab/DART-ID.
Author Summary Identifying and quantifying proteins in single cells gives researchers the ability to tackle complex biological problems that involve single cell heterogeneity, such as the treatment of solid tumors. Mass spectrometry analysis of peptides can identify their sequence from their masses and the masses of their fragment ion, but often times these pieces of evidence are insufficient for a confident peptide identification. This problem is exacerbated when analyzing lowly abundant samples such as single cells. To identify even peptides with weak mass spectra, DART-ID incorporates their retention time – the time when they elute from the liquid chromatography used to physically separate them. We present both a novel method of aligning the retention times of peptides across experiments, as well as a rigorous framework for using the estimated retention times to enhance peptide sequence identification. Incorporating the retention time as additional evidence leads to a substantial increase in the number of samples in which proteins are confidently identified and quantified.
