Peptidomics: Methods in the Search for Molecular Disease Signatures

Peptides play important roles in human physiology. They present as intact peptides or can arise from protein degradation. Regardless of the source, peptidomics—the characterization of intact peptides in a biological sample—is fast emerging as an approach to uncover another layer in human health and disease. Although challenges remain when sampling peptidomes, advances in sample processing, mass spectrometry, and data analysis have enabled the identification of peptide biomarkers for cancer and infectious disease diagnostics.

How are peptidomes produced?

Peptidomics follows a three-step procedure to generate a list of the peptides present in clinical samples, along with their abundances. First, sample processing inactivates and degrades proteins, leaving only the peptides for analysis. Then, mass spectrometry generates high-throughput data of all the molecules that comprise the peptides present in the samples. Finally, bioinformatics tools are used to identify and quantify the unique peptides in each sample.

Conventional approaches in peptidome generation adopt a “bottom-up” approach. Here, proteins are digested with an enzyme—typically trypsin—to generate peptide fragments to facilitate data analysis.¹ Advances in mass spectrometry have enabled a “top-down” approach, where intact peptides are directly ionized and fragmented inside the mass spectrometer.² This approach helps identify a peptide’s primary structure, peptide modifications, and degradation products for distinguishing peptide isoforms.³

Once the peptidomes have been generated, bioinformatics tools are used to discern peptide sequences and structures. Previous data analytics employ a data-dependent acquisition approach (DDA). In DDA, the mass spectrometer performs analyses on a subset of preselected precursor ions with specific characteristics above signal noise.⁴ More recently, the data-independent acquisition approach (DIA) interrogates all peptides within a defined ionic range, increasing the resolution for identifying low-abundance peptides.^5,6

Peptide-based biomarkers in disease

Advances in generating and analyzing peptidomes have helped identify intact peptide isoforms and their roles in human physiology. "Advances in mass spectrometry, such as SLIM-based high-resolution ion mobility, have helped characterize products of deamidation—a post-translational modification—that can impact drug efficacy or stability," explains James Atwood, Senior Vice President of ‘Omics at MOBILion Systems. Peptidomics has also helped identify degradation products in extracellular fluids. Tumor cells can use proteolytic mechanisms that modulate the expression and activity of adhesion proteins, releasing peptide fragments.⁷ Other peptide fragments from cleaved proteins can regulate other cellular phenotypes such as transcriptional regulation and cell adhesion.^8,9

Meeting the challenges of peptidomics—sample collection

The challenges of performing peptidomics analyses begin from sample collection. In top-down proteomics, the integrity of whole proteins must be maintained as much as possible through the entire processing procedure.¹⁰ “This also applies to peptidomics. Any sample processing procedure requires proper handling procedures to minimize peptide degradation before mass spectrometry analyses,” notes Michael Pisano, Executive Vice President of Proteomics at Discovery Life Sciences. He continues by saying, “While there will always be a lag time between sample collection and processing, ensuring that the processing protocol is consistent across all samples is more important.”

Meeting the challenges of peptidomics—peptidome generation

The ability to generate robust peptidomes also relies on the ability to detect the peptides and their isoforms. Previous mass spectrometers lack the separating power to identify peptide isoforms and post-translational modifications, especially for those with similar molecular weights.¹¹ In response to this, two companies have developed distinct technologies to improve the resolution for detecting peptide sequences.

Bruker developed one such approach with the timsTOF SCP mass spectrometer.¹² According to Pierre-Olivier Schmit, Business Development Manager of Proteomics, “The key to their technology’s success lies in the two trapped ion mobility separation (TIMS) device within the timsTOF SCP. The two TIMS components provide their own accumulation and separation steps to sequentially release ions with similar Collisional Cross Section (CCS) characteristics prior to a mass/charge-based separation in the Time of Flight (TOF) analyzer.”

MOBILion Systems also prepared an alternative approach, harnessing the ion path length to develop High-Resolution Ion Mobility Mass Spectrometry (HRIM-MS). Based on Structures for Lossless Ion Manipulation (SLIM), this mass spectrometer can differentiate between peptides of similar molecular weights. The serpentine layout that MOBILion adopts in its mass spectrometer enables the separation of ions with similar molecular weights by their ionic properties. To prevent the loss of any ions, electrodes are added to generate an electric field, which enhances molecular separations and moves ions without signal loss.

Meeting the challenges of peptidomics—bioinformatics

Being able to process multiple ‘omics datasets is also essential for elucidating the role that peptides play in human health and diseases. Chief among the priorities in data storage is the ability to maintain a robust data management system. There needs to be a data management and analysis platform to prevent manual naming convention translation errors and tracking peptide sequence metadata. Platforms like Paradigm4 are tackling this challenge head-on by placing experimental metadata, automated naming convention liftovers, and other ‘omics data into a single place,” says Zachary Pitluk, Vice President of Life Science Business Development at Paradigm4. The ability to consolidate this data will help streamline statistical analyses, identify mutations associated with peptide abundances, and identify novel biomarkers associated with disease outcomes.

Conclusions

Peptidomics provides an unprecedented opportunity to characterize peptide sequences and their post-translational modification. This is especially important when developing peptide-based medications or identifying isomeric species that contribute to health and disease. "Although conventional mass spectrometry pipelines cannot distinguish between peptide isomers, technological advances in ion mobility, including SLIM have provided the ability to unlock a huge fraction of the peptidome," Atwood explains. Furthermore, the ability to curate peptidomics data into cloud-based platforms can minimize transcription errors and centralize information about novel peptides into a single place. With further improvements on the way, the future of peptidomics for diagnostics and drug development is bright.

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