Advancing Biological Understanding Through Cutting-Edge Proteomics

By the Mass Spectrometry Resource Laboratory (MSRL) at the University of Rochester

Introduction

Proteins are essential for how cells, tissues, and organs function. They are involved in everything from signaling and metabolism to immune response and structural support. Changes in protein expression, localization, or function can reveal a lot about how biological systems work—whether in healthy states or when things go awry; understanding how proteins are regulated and how they interact with each other is key to gaining a better grasp of the biology that drives cellular activities and organismal development.

Proteomics, the systematic and comprehensive study of proteins, has become an invaluable tool for exploring the complexity of biological systems. While genomics focuses on the DNA or RNA sequences, proteomics dives into the dynamic world of proteins—how they’re made, folded, modified, interact, and degraded within the cell. These insights are essential for understanding how proteins contribute to normal cellular functions and how changes in these processes can offer new perspectives on biology.

The Mass Spectrometry Resource Laboratory (MSRL) at the University of Rochester uses cutting-edge technologies and expertise in proteomics to offer in-depth proteome profiling. Using high-resolution mass spectrometry, the facility enables researchers to explore biological systems with accuracy and precision. This paper will highlight the importance of proteomics in advancing our understanding of biology, while describing how the MSRL prepares, measures, and analyzes complex proteomic data.

Proteomics Sample Preparation: Challenges and Solutions

Proteins are highly dynamic and can be influenced by many factors, such as changes in the environment, disease processes, or cellular responses. One of the main challenges in proteomics is preserving proteins during sample collection, storage, and processing. Proteins are prone to degradation or modifications—like phosphorylation, acetylation, or ubiquitination—that can affect their behavior. To ensure accurate results, it’s important to minimize any sample manipulation and keep proteins as intact as possible.

At the MSRL, we use thoroughly tested and optimized protocols to extract proteins from biological samples while maintaining their original state. These methods are designed to recover a broad spectrum of proteins, from abundant structural proteins to low-abundance signaling molecules. We also focus on preserving critical post-translational modifications (PTMs), which are important for understanding how proteins function and interact. By carefully preserving these features, we ensure that our proteomic analyses reflect the true biological state of the sample.

High-Throughput Proteomic Analysis Using Mass Spectrometry

Proteomics requires advanced analytical tools that can measure a large variety of proteins and their modifications in a single sample. Tandem mass spectrometry (MS/MS) has become the go-to method for this, due to its sensitivity, precision, accuracy, and unbiased ability to identify proteins based on their mass-to-charge ratio (m/z). The MSRL uses state-of-the-art mass spectrometers that include the Thermo Orbitrap Astral and Bruker timsTOF Ultra that allow for accurate identification and quantitation of proteins.

The ability to perform high-throughput proteomic analysis is key to uncovering meaningful patterns in complex samples, such as tissue biopsies or body fluids, which contain thousands of proteins at different abundance levels. By coupling liquid chromatography (LC) with tandem mass spectrometry (LC-MS/MS), we can separate complex protein mixtures, which allows us to detect even rare proteins. This technology also enables us to analyze dynamic biological processes in real-time, such as changes in protein activity or protein interactions in response to different stimuli.

Proteomics is not just about identifying proteins—it’s also about understanding what those proteins are doing in the cell. LC-MS/MS allows us to analyze post-translational modifications (PTMs) that can significantly affect where proteins are localized or how they function. PTMs are pivotal in regulating protein behavior, and understanding these changes is crucial for uncovering how proteins interact with each other and contribute to cellular processes.

Data Interpretation and Bioinformatics Integration

The amount of data generated by high-throughput proteomic experiments can be overwhelming. A single experiment can produce millions of data points across multiple dimensions, and extracting useful biological insights from that data requires specialized computational tools. Depending on the goals of the experiment, data can be collected using different methods, like Data-Dependent Acquisition (DDA) or Data-Independent Acquisition (DIA), each offering specific advantages.

At the MSRL, we use a range of software tools to process raw data from mass spectrometry, identify proteins, and quantify their expression levels. We rely on tools like DIA-NN and Proteome Discoverer to analyze the data, localize protein modifications, and measure differences between samples. By combining these powerful tools, we’re able to uncover biological patterns and gain deeper insights into how proteins are working in different biological contexts.

Proteomics for Biological Insights

Proteomics provides a wealth of information that helps researchers understand how biological systems function. By profiling proteins—looking at their abundance, modifications, and interactions—proteomics reveals how biological processes are regulated at the molecular level. Changes in proteins can reflect alterations in cellular behavior, and proteomics allows us to study these changes systematically.

For example, proteomics has been used to examine proteins involved in fundamental processes like the regulation of the cell cycle, metabolism, or cell death. By studying these proteins in detail, scientists can gain a better understanding of how cells respond to environmental stimuli, stress, or internal signals. This can offer insights into how cells grow, differentiate, and adapt to changing conditions.

Proteomics also allows us to investigate how proteins interact with one another to form networks that drive cellular processes. Understanding protein-protein interactions is essential for getting a fuller picture of how cellular systems work together to maintain balance and respond to changes in their environment. These insights into protein function and interaction are key to understanding the bigger picture of biology.

Conclusion

Proteomics is a powerful tool for gaining insights into the molecular workings of biological systems. By profiling proteins—looking at their abundance, modifications, and interactions—proteomics allows researchers to understand how biological processes are regulated at the molecular level. This approach helps to reveal the complex network of interactions and regulations that underpin cellular functions and responses to various stimuli.

The University of Rochester Mass Spectrometry Resource Laboratory is committed to advancing biological research by providing the tools and expertise needed to explore the proteomic landscape. With our high-resolution mass spectrometry platforms and advanced data analysis capabilities, we are able to provide researchers with the insights they need to deepen our understanding of how proteins drive cellular behavior and contribute to broader biological functions.

For more information, and for consultations about your biomedical research project, visit proteomics.urmc.edu