Advances in Mass Spectrometry-Based Proteomics

The impact of mass spectrometry (MS) based proteomics on cell biological understanding is gaining new ground each day. Several recent advances in research and product development are now helping drive the field into the exciting new world of single-cell proteomics and beyond.

The promise of single-cell proteomics

Proteomics involves the characterization of the full complement of proteins within cells. Such insight can shed light on cellular processes, cell states, the appearance of disease markers, and other important details. The growing field of single-cell proteomics (sc-proteomics) narrows the focus toward biological activities and functions within individual cells. While proteomics aims to characterize protein levels in cells at the population level, sc-proteomics strives to uncover distinct qualitative and quantitative differences among individual cell types and states. This promises to inform a higher understanding of biological function, dysfunction, and the effects of therapeutic intervention.

Scaling down proteomics

Much of the proteomics successes thus far have involved populations of cells. There are significant challenges, however, in scaling proteomics investigations from cell population levels down to the single-cell level.

Technologies such as multi-parameter fluorescence-assisted cell sorting (FACS), originally developed for the cell culture field, have been useful in the proteomic analysis of small groups of cells. Other technologies such as single-cell mass cytometry (CyTOF) have shown the capability of detecting small numbers of proteins in single cells. Although valuable, these approaches have proven to be limited in sensitivity, proteome coverage, and the throughput needed to analyze single cells and replicates on a practical time scale.

Mass spectrometry single-cell (sc) proteomics

Advanced MS technologies have emerged that address these limitations. New instruments are capable of attomole (10 -18) sensitivity, within the range of single-cell protein levels, detecting thousands of proteins in a single run using multiplexed sample analysis.

Sample preparation

As an important initial step in MS analysis, sample preparation techniques must provide sufficient starting materials to account for losses due to handling, absorption, and background interferences in samples. Separation performance is another critical parameter required to remove background interferences and optimize peptide isolation and detection. These considerations are especially relevant to sc-proteomics and the need to identify distinct differences within small-volume samples.

Advances in technology and product development are paving the way to better accuracy, precision, throughput, and overall quality of MS-based sc-proteomics investigations.

Sample enrichment

A growing number of novel sample enrichment technologies, such as that originally pioneered by Claudia Ctortecka, PhD, and colleagues at the Research Institute of Molecular Pathology in Vienna, Austria, use automated single-cell isolation coupled with picoliter dispensing workflows. Samples are deposited on microscope slide-sized chips, typically in multi-arrays wells. After enrichment and labeling, samples are then prepped for MS analysis. These novel devices eliminate manual handling and evaporation, allowing direct injection of single-cell samples via a standard LC-MS/MS autosampler. Automating the sample handling process increases reproducibility, while the multiplexing of sample enrichment enables analysis of up to hundreds of single cells and quantification of thousands of proteins in a single analysis.

Low-flow nano LC

As mentioned, separation performance is vital in removing background interferences and allowing clarity and reproducibility in downstream analysis. New nanoflow UHPLC technologies are designed to deliver the low flow rates (10 nL/min) and the high pressures (up to 1500 bar or more) required by the latest high-resolution columns. By eliminating dead volumes and optimizing flow, these new nanocolumns can achieve high-sensitivity detection of peptides and proteins that are present at vanishingly low levels, such as those present in sc-proteomics samples. A range of column chemistries and scaffolds, such as polymeric and nanofabricated substrates, enable column scouting, high-fidelity separations, and deep sc-proteome coverage.

Protein quantitation

Quantitation is another challenge in single-cell analysis, particularly for scaling up and multiplexing sc-proteomic sample replicates. To this end, the field has leveraged the use of isobaric tags. These are molecules that can be covalently attached to peptides to produce identical masses that fragment inside the mass spectrometer to produce different-sized ions. This technique allows peptides to be tagged without changing their initial masses and subsequently quantified upon LC-MS/MS fragmentation. Commercialized as TMTs (short for tandem mass tags), the number of possible tagging combinations was recently expanded to 18, allowing multiplexing for three replicates of six samples in a single experiment.

Data acquisition and analysis

Data acquisition and data quality are big challenges for sc-proteomic investigations as well. MS systems must deliver sensitivity and accuracy on a per-sample basis, while delivering the precision and throughput necessary to analyze multiple sample replicates.

These challenges are being met by the latest generation of hybrid mass spectrometers and data analysis technologies. Traditional data-dependent acquisition (DDA) of ultra-low input samples suffers from the accumulation of missing values (missing peptides) as the size of the sample cohort increases. Data-independent acquisition (DIA) methods are not fully compatible with isotope-encoded (TMT) sample multiplexing. Novel approaches are implementing concepts like identification-independent data analysis tools to compare TMT proteome signatures across hundreds of samples and different cell types. These approaches are working to increase quantitative protein coverage while enhancing data reproducibility. 

Outlook

Advances in technology and product development are paving the way to better accuracy, precision, throughput, and overall quality of MS-based sc-proteomics investigations. New MS instruments, in combination with the latest quantitative labels and analysis techniques, have the power to analyze over 300 cells per day, cutting the total analysis time of 10,000 cells to just over one month. 

With this growing cache of advancements and accelerated workflows, the focus can now broaden to include new territory. MS-based spatial-omics aims to use sc-proteomics to deduce discrete differences in intact tissue. Although laser-capture microdissection and other techniques coupled with mass spectrometry have been developed, none have achieved single-cell resolution. Single-cell resolution of intact tissue is a new frontier for the field of MS-based sc-proteomics and it’s now within view.