A New Era of MS-Based Proteomics To Advance Human Wellness

Human health research is a vast space that requires a multidisciplinary effort. A commonality between the wide range of disciplines involved in human health is the use of mass spectrometry (MS), which has evolved to become a workhorse for research in this field. MS is a highly established technique for analyzing proteins and peptides, and it has applications in proteomics that include cataloging protein expression, identifying protein modifications and characterizing protein–protein interactions.

Modern advancements in MS have enabled the use of proteomics in more biological fields than ever before. MS-based proteomics offers the unique advantage of enabling comprehensive analysis of proteins and their changes, including post-translational modifications (PTMs), to provide a global interpretation of biological systems. One MS-based method that has been gaining significant traction recently is data-independent acquisition (DIA). Introduced over a decade ago, DIA has become a method of choice for next-generation proteomics workflows.

Technology Networks had the pleasure of speaking to Katherine Tran, Senior Manager, Global Strategic Marketing – Life Sciences Research at SCIEX, to find out how DIA-MS works and how it can be used to further our understanding of the complex human proteome.

Katie Brighton (KB): Can you tell me a little about why analyzing less abundant species of protein might be important in the pursuit of biological insights?

Katherine Tran (KT): Interestingly, the proteins of lesser abundance are usually the ones of most biological interest or concern, especially in human health. For example, many diseases, such as cancer, do not show obvious clinical symptoms until the condition is in advanced stages and difficult to treat successfully. However, if we can detect these diseases in earlier stages by finding their protein biomarkers as early as possible, we can reduce disease-related mortality by optimizing treatment plans by knowing what treatment to apply to which individual and when.

Unfortunately, this is easier said than done. The vast dynamic range of human bodily fluid proteomes, such as those in blood plasma, makes it difficult to detect these protein biomarkers, especially at very low abundances. Like discovering a needle in a haystack, many important signalling or regulatory proteins have concentrations in the microgram per milliliter to picogram per milliliter range, where protein concentrations can differ by over 10 orders of magnitude in the blood. In addition, proteins of high abundance, such as globulin, or coagulation factors in the range of grams per milliliter often mask lower abundant proteins, which further complicates the detection of these biomarkers. As a result, proteomics researchers continue to pursue advancements in analytical techniques and methods that can enhance the detection of trace levels of sensitivity and specificity.

KB: What is DIA-MS, and how is it applied within quantitative proteomics?

KT: In tandem mass spectrometry (MS/MS), DIA allows all detected ionizable precursor ions to be further fragmented in the activation cell regardless of abundance or other criteria. Not only does this improve reproducibility between sample sets, but we can also expect a virtually complete, comprehensive data set containing fragment data for all precursor ions, including low abundant protein species.

In contrast to its preceding method, data-dependent acquisition (DDA), DIA generally uses wider precursor selection windows for MS/MS that allow multiple compounds through the mass analyzer simultaneously. These windows are stepped across the entire precursor mass range so that all precursor masses are fragmented for every cycle. However, now that all detected ionizable precursors are selected for fragmentation, the spectra generated by DIA tend to be more complex than DDA-generated spectra.

To enhance the depth and specificity of DIA workflows, SCIEX and a group of highly recognized scientists proposed a unique DIA method called SWATH DIA, which uses many windows to minimize the number of precursor ions within each window. The combination of SWATH DIA and fast MS/MS scanning from time-of-flight (TOF) instruments allows iteration through all windows within each cycle while still covering a broad mass range. In addition, SWATH DIA allows variable window widths to further increase selectivity. With variable windows, the width of each precursor window is adjusted according to the complexity of the data found within that mass range. Very narrow windows are used where analyte density is greatest, and wider windows are used where analytes are more sparsely populated. This increases the percentage of high-quality identifiable and quantifiable precursors.

KB: Recently, SCIEX launched Zeno SWATH DIA, which harnesses the power of the Zeno trap for SWATH DIA. Why is this important to the data acquisition method?

KT: The Zeno trap itself is also a novel technology that is exclusively available on our latest mass spectrometer, the ZenoTOF 7600 system, where it creates a trap and release setup within the ion path to increase the duty cycle and boost MS/MS sensitivity.

The duty cycle refers to the percentage of ions that are injected into the TOF region of the mass spectrometer. However, this is typically only around 5–25% of the ions in standard TOF instruments. To overcome this challenge, SCIEX introduced the Zeno trap, which gates the precursor ions once they enter the mass analyzer. Once all the ions have entered, they are released in order from higher mass-to-charge ratio to lower mass-to-charge ratio, and all arrive at the TOF accelerator at the same time. Therefore, the Zeno trap increases the duty cycle to greater than 90%.

Since the goal of DIA is to create a virtually complete and comprehensive dataset, the Zeno trap further enhances this workflow.

KB: What are the major challenges in proteomics research, and how is Zeno SWATH DIA designed to address these challenges?

KT: While current MS-based proteomics methods have focused on increasing the number of proteins identified in a given sample, many biological and biomedical questions require the processing of large sample series along with the quantification of those proteins. We need to determine the activities, interactions, and proteoforms of the proteins to provide practical biological insight. However, high-throughput workflows and accurate quantitative analyses often pose major challenges in proteomics. Zeno SWATH DIA was designed to address these challenges. By combining the higher duty cycle of the Zeno trap with the fast speeds of SWATH DIA, we can maximize the number of analytes measured with increased sensitivity and within a faster time frame.

KB: What does the workflow look like when using Zeno SWATH DIA, and how does this compare to traditional protein quantification methods?

KT: One benefit of applying Zeno SWATH DIA to proteomics research is that researchers can use significantly less sample amounts while still gaining better protein coverage than traditional methods. We have even analyzed samples in sub-nano amounts using Zeno SWATH DIA, which is particularly important as we move toward this single-cell movement.

In addition, the combination of the unique technologies and methods that make up Zeno SWATH DIA offers researchers a more in-depth proteomic snapshot of a given sample set. Improved reproducibility due to the reduction of missing values and better MS/MS fragmentation will increase protein and peptide quantification accuracy. In an internal study, a highly multiplexed targeted peptide quantification assay was developed to explore the quantitative capability of Zeno MS/MS on the ZenoTOF 7600 system. Using a mixture of 804 heavy labeled synthetic peptides dosed into plasma, the increase in peptide sensitivity due to Zeno MS/MS was found to be almost 6 fold. Excellent reproducibility was also observed with a median CV of 6.1% across all 804 peptides. Furthermore, concentration curves were generated to explore the sensitivity in digested plasma, and the median lower limit of quantification (LLOQ) for the peptides was found to be 207 amol on column.

KB: What are the applications of Zeno SWATH DIA, and how does it benefit researchers in these fields?

KT: Although we have been discussing how Zeno SWATH DIA can be applied to the research of human health, there are a wide variety of applications for this technique. Although DIA-MS has been used predominantly in proteomics, it has been extended into metabolomics and lipidomics, biopharmaceuticals, clinical and forensic research, and even food and environmental sciences.

Using Zeno SWATH DIA, researchers can increase the sensitivity of their mass spectral data—in the depth of coverage and quality of fragmentation—and increase the speed of data acquisition. For more information, visit sciex.com/ZenoSWATHDIA and download an exclusive white paper about Zeno SWATH DIA here.

Katherine Tran was speaking to Katie Brighton, Scientific Copywriter for Technology Networks.