Towards single-cell LC-MS phosphoproteomics

Abstract

Protein phosphorylation is a ubiquitous posttranslational modification, which is heavily involved in signal transduction. Misregulation of protein phosphorylation is often associated with a decrease in cell viability and complex diseases such as cancer. The dynamic and low abundant nature of phosphorylated proteins makes studying phosphoproteome a challenging task. In this review, we summarize state of the art proteomic techniques to study and quantify peptide phosphorylation in biological systems and discuss their limitations. Due to its short-lived nature, the phosphorylation event cannot be precisely traced in a heterogonous cell population, which highlights the importance of analyzing phosphorylation events at the single cell level. Mainly, we focus on the methodical and instrumental developments in proteomics and nanotechnology, which will help to build more accurate and robust systems for the feasibility of phosphorylation analysis at the single cell level. We propose that an automated and miniaturized construction of analytical systems holds the key to the future of phosphoproteomics; therefore, we highlight the benchmark studies in this direction. Having advanced and automated microfluidic chip LC systems will allow us to analyze single-cell phosphoproteomics and quantitatively compare it with others. The progress in the microfluidic chip LC systems and feasibility of the single-cell phosphoproteomics will be beneficial for early diagnosis and detection of the treatment response of many crucial diseases.

Using Dicationic Ion-Pairing Compounds To Enhance the Single Cell Mass Spectrometry Analysis Using the Single-Probe: A Microscale Sampling and Ionization Device

“…12–14 MS is a versatile technique to simultaneously analyze a large number of molecules in a short period of time. Traditional MS approaches to cell analysis were restricted to a population of cells (such as cell lysate), where an averaged result is obtained.…”

Understanding cell signaling in cancer stem cells for targeted therapy – can phosphoproteomics help to reveal the secrets?

“…In addition to the requirement of sophisticated and state-of-the-art technologies, also the dynamic nature of phosphorylation requires careful avoidance of enzymatic or chemical dephosphorylation by means of phosphatase inhibitors, making the analysis a challenging task [90]. Phosphorylation events are time dependent and thus phosphoproteomics can only provide a snapshot of the particular condition.…”

“…Until now, there are still technical limitations to perform phosphoproteomics at the single-cell-level, with sensitivity being the primary constraint [90]. For a comprehensive state-of-the-art phosphoproteomics approach the protein amount of a single cell is too low.…”

Synthesis of BC@mTiO2 hybrid nanofibers for highly efficient enrichment and detection of phosphopeptides

“…Sensitive detection of phosphorylated proteins is required for understanding the disease mechanisms and the development of early diagnostic technologies. Mass spectrometry (MS) has become an indispensable tool for analysis of various biomolecules including phosphoproteins or phosphopeptides (Polat and Ozlu 2014;Leitner et al 2011;Chambers et al 2012;Jünger and Aebersold 2014;Gong et al 2014;Lin et al 2016; Thompson et al 2013). However, phosphorylated proteins are frequently masked by a large number of non-phosphorylated proteins or other biomolecules inside the cells.…”

“…Among these, protein phosphorylation is widely involved with the regulation of many cellular processes including the signal transduction, gene expression, cell cycle, growth and apoptosis (Shortreed et al 2015;Olsen et al 2006;Polat and Ozlu 2014). Inappropriate phosphorylation of proteins invariably leads to disordered cellular processes, which are mostly the direct reasons for many serious diseases (Maceyka et al 2012).…”