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). DART-ID implements principled Bayesian frameworks for global retention time (RT) alignment and for incorporating RT estimates towards improved confidence estimates of peptide-spectrum-matches. When applied to bulk or to single-cell samples, DART-ID increased the number of data points by 30-50% at 1% FDR, and thus decreased missing data. Benchmarks indicate excellent quantification of peptides upgraded by DART-ID and support their utility for quantitative 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://dart-id.slavovlab.net.

Transcriptional and post-transcriptional regulation shape tissue-type-specific proteomes, but their relative contributions remain contested. Estimates of the factors determining protein levels in human tissues do not distinguish between (i) the factors determining the variability between the abundances of different proteins, i.e., mean-level-variability and, (ii) the factors determining the physiological variability of the same protein across different tissue types, i.e., across-tissues variability. We sought to estimate the contribution of transcript levels to these two orthogonal sources of variability, and found that scaled mRNA levels can account for most of the mean-level-variability but not necessarily for across-tissues variability. The reliable quantification of the latter estimate is limited by substantial measurement noise. However, protein-to-mRNA ratios exhibit substantial across-tissues variability that is functionally concerted and reproducible across different datasets, suggesting extensive post-transcriptional regulation. These results caution against estimating protein fold-changes from mRNA fold-changes between different cell-types, and highlight the contribution of post-transcriptional regulation to shaping tissue-type-specific proteomes.

Analyzing proteins from single cells by tandem mass spectrometry (MS) has recently become technically feasible. While such analysis has the potential to accurately quantify thousands of proteins across thousands of single cells, the accuracy and reproducibility of the results may be undermined by numerous factors affecting experimental design, sample preparation, data acquisition and data analysis. We expect that broadly accepted community guidelines and standardized metrics will enhance rigor, data quality and alignment between laboratories. Here we propose best practices, quality controls and data-reporting recommendations to assist in the broad adoption of reliable quantitative workflows for single-cell proteomics. Resources and discussion forums are available at https://single-cell.net/guidelines .

To mark the 15th anniversary of Nature Methods, we asked scientists from across diverse fields of basic biology research for their views on the most exciting and essential methodological challenges that their communities are poised to tackle in the near future.