Single cell protein analysis for systems biology

Abstract

<jats:p>The cellular abundance of proteins can vary even between isogenic single cells. This variability between single-cell protein levels can have regulatory roles, such as controlling cell fate during apoptosis induction or the proliferation/quiescence decision. Here, we review examples connecting protein levels and their dynamics in single cells to cellular functions. Such findings were made possible by the introduction of antibodies, and subsequently fluorescent proteins, for tracking protein levels in single cells. However, in heterogeneous cell populations, such as tumors or differentiating stem cells, cellular decisions are controlled by hundreds, even thousands of proteins acting in concert. Characterizing such complex systems demands measurements of thousands of proteins across thousands of single cells. This demand has inspired the development of new methods for single-cell protein analysis, and we discuss their trade-offs, with an emphasis on their specificity and coverage. We finish by highlighting the potential of emerging mass-spec methods to enable systems-level measurement of single-cell proteomes with unprecedented coverage and specificity. Combining such methods with methods for quantitating the transcriptomes and metabolomes of single cells will provide essential data for advancing quantitative systems biology.</jats:p>

DART-ID increases single-cell proteome coverage

“…Recently, we have developed Single Cell Proteomics by Mass Spectrometry (SCoPE-MS) that allows quantifying proteins in single mammalian cells [1]. One of the challenges for SCoPE-MS is identifying the sequences of lowly abundant peptides since they may generate only a few fragment ions that are insufficient for confident identification [2,3]. Such low confidence peptides are generally not used for protein quantification, and thus reduce the data points available for further analyses.…”

Automated sample preparation for high-throughput single-cell proteomics

“…Minimizing sample losses and maximizing throughput is a major requirement for applying ultrasensitive MS to biological problems 3,8 . It has motivated many colleagues to develop sample preparation methods with minimal volumes and low cleanup losses 2,6 .…”

“…Before applying mPOP to prepare and analyze single-cell proteomes, we sought to estimate the instrument measurement noise in the context of SCoPE-MS sets. This estimate is motivated by our concern that factors unique to ultra-low abundance samples, such as counting noise 3,8 , may undermine measurement accuracy. To isolate the noise in instrument (Q-exactive) measurement from noise due to biological variation and sample preparation, we used mPOP to prepare a 100×M…”

“…Methods for preparing sub-microgram protein samples for LC-MS/MS often use sophisticated custom-made equipment [1][2][3] and generally lyse cells by detergents or chaotropic agents like urea 3,4 .…”

Multi-Omics Approach: New Potential Key Mechanisms Implicated in Cardiorenal Syndromes

“…Proteins analysis and quantification have been completely transformed and modified by mass spectrometry (MS)-based methods. MS is a revolutionary method adapted for high-throughput analyses of thousands of proteins in cells or body fluids [80-82]. Recent technological advances in proteomics have allowed a wide-scope analysis of protein patterns in bodily fluids, allowing the identification of numerous protein markers in various conditions.…”