The Evolution of Mass Spectrometry in Proteomics: From Quantification to Structural Analysis

By Dr. Priyom Bose, Ph.D.Reviewed by Lily Ramsey, LLM

Mass spectrometry (MS) is an indispensable analytical tool in proteomic research. It is used to determine protein interactions, catalog protein expression, and detect sites of protein modification.¹ This article focuses on the evolution of MS that has enriched our understanding of different aspects of proteins. 

​​​​​​​​​​​​Image Credit:S. Singha/Shutterstock.com 

Introduction of MS in Proteomic Research

Genes encode proteins that are essential to carry out life processes.² Initially, proteomic research was focused on linking specific proteins to biological functions. It was soon understood that a single protein may not be necessarily associated with one function or physiology. The main challenge of proteomics is to uncover the diverse roles of proteins in cells.³

Large-scale proteomic studies have enabled the identification of proteins' locations in cells, protein-protein interactions, and post-translational modification sites present in amino acid sequences. Elucidating all protein modifications and sequence variations is important to understanding the impact of protein modifications on biological functions. 

Conventionally, protein structures were studied using cryogenic electron microscopy (cryo-EM), X-ray crystallography, and nuclear magnetic resonance (NMR), which are mostly performed in vitro.⁴ In contrast, MS enabled analysis of native proteins and native protein complexes.⁵ Recently, this tool has also been used to analyze entire cells for measuring the state of protein folding within complete proteomes.

Advancements in MS have enabled intact protein analysis. For intact protein identification, fragment ion data at amide linkages is important for the entire protein backbone. This strategy has not only helped identify proteins but also accurately detected the position of sequence variation. 

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Evolution and Technological Advancements in MS for Proteomics

MS was invented during the hunt for the electron, and it has undergone significant advancements over time. The first mass spectrometer was developed by J.J. Thomson, a British physicist.⁶ In the twentieth century, MS was widely used in analytical chemistry. Owing to its inability to ionize and vaporize labile molecules such as proteins, MS was mostly used for molecular biology research. However, the introduction of the electrospray ionization technique revolutionized MS applications.⁷ 

In 1988, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was developed which became an important tool for analyzing biological samples.⁸ In the 1990s, this tool was used to study protein structures. In 1992, low-level peptide analyses were conducted using MS techniques.⁹

The electrospray ionization method was used to design the liquid chromatography (LC)–MS setup, which was able to separate femtomole amounts of peptides from complex mixtures. This method proved invaluable for detecting peptides present in the immune system, particularly antigen presentation by major histocompatibility complex (MHC) molecules. The development of an electrospray nano-LC–MS/MS platform linked to a comprehensive database has become the heart of MS-based proteomics.

The invention of high-resolution and high-mass accuracy instruments, particularly Orbitrap and time-of-flight (TOF) mass analyzers, stems from the massive drive to identify more peptides and post-translational modifications.¹⁰ The massive improvements in mass resolution in recent instruments have generated interest in top-down proteomics studies. Previously, scientists experienced immense challenges in carrying out similar experiments because of the need for expensive high-field magnets for ion cyclotron resonance MS of intact proteins.

The use of hybrid instruments associated with the utilization of different ion analyzers or separators has significantly improved MS capabilities. For instance, the development of the triple quadrupole MS brought about significant analytical improvements. Here, the first quadrupole was used to select m/z values, the second was used as a collision cell, and the third was used to perform a more routine analysis of ions.¹¹ The latest Orbitrap Fusion Lumos Tribrid mass spectrometer includes five different ion separation/storage devices.

The use of electron transfer dissociation (ETD) and ultraviolet photodissociation (UVPD) methods for efficient fragmentation of intact proteins has significantly improved the prospects for top-down MS.¹² The ion routing multipole has also been implemented for higher-energy collisional dissociation (HCD) ion fragmentation. The key advantages of this integration entail simultaneous experiments within the instrument, which accelerated scan speed and consequently increased the number of tandem mass spectra collected for peptide ions.

A trapped ion mobility spectrometer (TIMS) uses electric and radiofrequency fields to trap ions in a flowing gas. Based on the same concept, the parallel accumulation-serial fragmentation (PASEF) method was developed, which is associated with a mass selective release of peptide ions from the TIMS device for MS/MS. Recently, TIMS and PASEF methods have been combined to increase the number of tandem mass spectra of peptides and the number of identified proteins.¹³

Currently, user-friendly software programs and servers are available that can easily and automatically integrate the large datasets generated by structural proteomics experiments and process them in machine learning models. Many scientists are currently using this method to determine protein structure in a complex environment of cells or tissues. Artificial intelligence models, such as Alphafold and RoseTTAfold, have exhibited immense promise for predicting protein structures.¹⁴

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Challenges and Future Outlooks

The continual technological and methodological improvements in MS have advanced the field of proteomics. Implementation of these advancements could significantly enhance the diagnosis of medical conditions.

Despite MS's advantages in proteomics analysis, there are many challenges associated with it. A major challenge encountered by scientists while performing tandem MS for protein identification is the use of protein sequence databases that may not contain multiple alternative splice isoforms. Consequently, multiple bona fide protein products of alternative splice isoforms could not be identified because the target sequence was absent in the database. 

The absence of protein sequences in the protein database could also lead to incorrect peptide or protein identification. A large number of proteins are annotated in different places and this lack of standardized approach contributes to inaccurate protein identification.

In some cases, the same proteins bear different names and IDs in different databases used for MS/MS protein search. Therefore, the absence of standard protein identifiers increases the difficulty of accurate protein identification.  These challenges must be addressed in the future to improve the application of MS in proteomic research.

References

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10. Strader MB, Alayash AI. Exploring Oxidative Reactions in Hemoglobin Variants Using Mass Spectrometry: Lessons for Engineering Oxidatively Stable Oxygen Therapeutics. Antioxid Redox Signal. 2017;26(14):777-793. doi:10.1089/ars.2016.6805
11. Yost RA. The triple quadrupole: Innovation, serendipity and persistence. J Mass Spectrom Adv Clin Lab. 2022;24:90-99. doi:10.1016/j.jmsacl.2022.05.001
12. Yates JR 3rd. Recent technical advances in proteomics. F1000Res. 2019;8:F1000 Faculty Rev-351. doi:10.12688/f1000research.16987.1
13. Meier F, Park MA, Mann M. Trapped Ion Mobility Spectrometry and Parallel Accumulation-Serial Fragmentation in Proteomics. Mol Cell Proteomics. 2021;20:100138. doi:10.1016/j.mcpro.2021.100138
14. Perrakis A, Sixma TK. AI revolutions in biology: The joys and perils of AlphaFold. EMBO Rep. 2021;22(11):e54046. doi:10.15252/embr.202154046

Further Reading 

• How is Tandem Mass Spectrometry Used in the Life Sciences?
• MALDI-TOF Mass Spectrometry in the Biosciences
• Current Challenges in Mass Spectrometry Instruments
• Monitoring Doping using Mass Spectrometry