Advances in microscale separations towards nanoproteomics applications

Highlights

• Nanoproteomics represents a daunting analytical challenge in terms of sensitivity.

• NanoLC and CE coupled with MS can provide single cell level sensitivity.

• Front-end sample processing is the current bottleneck of overall sensitivity in MS proteomics.

• Significant advances have achieved towards nanoproteomics measurements.

• Further advances are still required for enabling broad nanoproteomics applications.

Abstract

Microscale separation (e.g., liquid chromatography or capillary electrophoresis) coupled with mass spectrometry (MS) has become the primary tool for advanced proteomics, an indispensable technology for gaining understanding of complex biological processes. In recent decades significant advances have been achieved in MS-based proteomics. However, the current proteomics platforms still face an analytical challenge in overall sensitivity towards nanoproteomics applications for starting materials of less than 1 μg total proteins (e.g., cellular heterogeneity in tissue pathologies). Herein, we review recent advances in microscale separation techniques and integrated sample processing strategies that improve the overall sensitivity and proteome coverage of the proteomics workflow, and their contributions towards nanoproteomics applications.

Introduction

Proteins are the workhorses of the cell, impacting nearly all aspects of cellular processes. The proteome by its nature is dynamic, and the acute state of the proteome (i.e., the proteotype) depends on both the genotype and external perturbations [1], [2]. Therefore, quantitative analysis of the dynamics of the proteome including post-translational modifications (PTMs) and its connection to phenotypes (e.g., diseases) has become indispensable in biological and clinical research [3], [4], [5]. Recent advances in mass spectrometry (MS)-based proteomics for both global deep-profiling of the proteome and selected types of PTMs (e.g., phosphorylation) [6], [7], [8] and targeted quantification of proteins from specific signaling pathways [9], [10] have greatly expanded our capabilities in performing proteogenomics and systems biology studies for gaining detailed mechanistic insights into physiological and pathological processes.

During the past decade, major advances have been achieved in nearly all areas of the proteomics workflow such as high resolution microscale chromatographic separations, mass spectrometry instrumentation, and bioinformatics data analysis tools to enable large-scale proteome interrogation [11]. Current state-of-the-art MS-based proteomics platforms can afford deep coverage for both the global proteome and selected PTMs in cell or tissue samples. For example, recent studies have reported the identification or quantification of ∼10,000 proteins [6], [7], >20,000 phosphorylation [12], [13] and >15,000 ubiquitination sites [12], [14].

Despite recent advances in improving overall proteome coverage, the current proteomics workflows typically require relatively large amounts of starting materials on the order of millions of cells or 100 s μg of proteins, which excludes many important biological and biomedical applications. The ability to effectively analyze extremely small amounts of protein samples (e.g., nanograms of proteins) from cells or tissues by MS is one of the most significant challenges for current MS-proteomics. Herein, we define sample amounts with less than 1 μg of total protein as “nanoscale” and proteomics analyses of these nanoscale samples as “nanoproteomics” (Fig. 1). The biomedical need for nanoproteomics technologies are compelling, including the analyses of tissue substructures, cellular microenvironments of disease pathologies, rare or small subpopulations of cells, extracellular vesicles, as well as single cell resolution profiling (Fig. 1). Some of these sample types are readily produced by existing technologies such as fluorescence activated cell sorting (FACS) [15], laser capture dissection (LCM) [16], [17], and exosome isolation techniques [18]. Moreover, single cell resolution genomics technologies, such as single-cell genomic sequencing [19] and single-cell transcriptomic profiling (RNA-Seq) [20], [21], have been making tremendous impact in biological research. However, the current state-of-the-art in MS-based proteomics still falls far short of the sensitivity required for single cell analyses.

Considerable efforts have been devoted to enhance the overall sensitivity of MS-based proteomics workflow towards enabling analysis of small samples, including the front-end sample processing, microscale separations, and MS instrumentation. Herein, we review recent advances in microscale separation, as well as nanoscale sample processing systems for proteomics analysis. Our focus will be on bottom-up proteomics, and the other important advances in top-down proteomics (measurement of intact proteins) [22] are not covered here.