Dr. Tim Veth, a postdoctoral researcher working with Prof. Nick Riley at the University of Washington, is tackling one of proteomics’ most complex challenges: deciphering the intricate world of glycoproteomics, where a single protein can exist in thousands of different forms. Throughout his journey from economics to mass spectrometry, his work is increasingly opening new pathways for cancer immunotherapy and antiviral treatments. Now, he invites all researchers to “join the battle” and dive even deeper into glycopeptides.

From economics to mass spectrometry

Tim’s journey into glycoproteomics wasn’t a straight line. Starting with a Bachelor’s in innovation sciences, which was primarily economics-based, Tim had no research or lab experience until his final year. That changed when he attended lectures by two professors discussing biotech and medicine development.

“One was mainly focused on making vaccines, and the other was focused on making cheaper alternatives for low- and middle-income countries,” Tim recalls. “I was really diving into the biotech behind this, and it was way more interesting than simply innovation sciences.”

This revelation led Tim to pivot his career path toward a master’s in drug innovation, where he learned to apply innovations to developing new medicines. His first internship, supervised by Richard Scheltema and Enrico Mastrobattista, introduced him to the world of mass spectrometry through research using cross-linkers for structural determination of protein complexes.

“That introduced me to the world of mass spec. Now, I couldn’t imagine doing something else,” he says.

Following a second internship in metabolomics-focused mass spectrometry, Tim pursued a PhD in a lab focused on phosphorylation-driven signalling in cancerous cells, cementing his place in the field of proteomics.

The interface of technology and biology

Tim’s move to Seattle for his postdoc happened almost by chance, meeting his current PI at a conference, but their research and personal alignment was perfect.

“I knew I liked two things: I liked to dive into the technology that mass spectrometry has to offer and how to improve it. But I also liked biology, and I wanted to be at the interface of technology and biology.”

His current work bridges these two interests, focusing on glycoproteomics, the study of proteins modified with complex sugar structures called glycans. This field presents unique challenges that Tim initially underestimated while his research was more focused on phosphoproteomics.

“I heard that glycoproteomics is challenging, but I didn’t really believe it,” he admits. “I thought, ‘How hard can it be? Just inject it into the mass spec and see what you get.’ But it doesn’t work like that. It’s way more complex than I thought.” It doesn’t help that mass spectrometry doesn’t give you a complete picture. “From a full puzzle, you only get like 25% or 30%. You know what you see, but you never know what you don’t see. That’s why you need different techniques.”

The complexity of glycoproteoforms

Tim’s perspective changed when he started working on a protein with 16 O-glycosylation sites and two N-glycosylation sites. What seemed like a straightforward analysis turned into a deep dive into extraordinary complexity.

“There were so many different glycoproteoforms of this one single protein that we couldn’t even analyse it using intact mass spectrometry. There were so many signals. Everything was immeasurable,” he explains.

The challenge stems from the sheer number of possible glycan combinations. A single protein might have thousands of different glycoproteoforms, many with isobaric masses, meaning they have the same mass but different glycan structures attached.

“Even by knowing the intact mass, we wouldn’t really resolve what glycoproteoform it belonged to. It really shows how deep you have to dive to know what these proteoforms are actually for.”

Technical advances and exponential growth

The field of glycoproteomics has seen exponential growth in technological capabilities. “In the early 2000s, glycoproteomic studies found maybe 10 to 30 different glycopeptides. A recent paper found over 100,000 different glycopeptides in one study, which showcases the increasing accessibility of understanding the biology behind glycosylation.”

This progress stems from improvements in method development, sample preparation, and analysis tools. Yet challenges remain, particularly in data analysis, a bottleneck Tim has encountered firsthand.

“The mass spec is pretty solid in collecting the data, but it doesn’t do analysis for you,” he notes. “There are quite a bit of software to help you identify glycopeptides, but the false discovery rate is really high, so you need a lot of manual verification.”

This analytical challenge extends to the laboratory workflow as well. In his search for reliable performance, Tim found that equipment quality can significantly impact experimental outcomes.

“We were looking for reliable columns, and people recommended IonOpticks columns,” he explains. “The first column lasted for many months, which is really impressive, and the performance was great. It also saved us the hassle of making our own in-house columns.” For Tim, this reliability translates directly into research productivity.

You can read more about developments in glycoproteomics in Tim’s perspective piece, and read about how architectural changes to the Tribrid MS platform, combined with Aurora Series columns, benefit glycoproteomic experiments in his recently published paper.

A balanced approach to research

When asked about overcoming the data analysis challenges in glycoproteomics, Tim’s advice is straightforward: “Learn how to code. Nobody will do it for you.” He emphasises that coding skills allow researchers to tackle novel problems creatively and efficiently. “Once you learn how to code, it expedites the whole scientific workflow and will really help you in your projects. Then you’ll be able to do stuff that nobody else can do, or has done before, because it’s a creative process.”

Tim also advocates for open science principles. “If you write your code in a way that people can use it too, you can start to create a stable platform. It will be helpful for other people and for yourself. I think it’s important in research that, whatever you make, make it accessible for others. The net benefit will be as big as possible.”

Tim’s approach to research balances technological development with biological exploration.

“I try to divvy up my projects between the mass spec technology side and the biology side, because they both have their advantages and disadvantages,” he explains. “Working with the mass spec, usually experiments are pretty quick. On the biology side, usually we need more patience. Sometimes you need to overexpress proteins in the cell line, or you need cells to do something specific, and that takes time.”

This balance keeps his work engaging and productive, allowing progress on one front while waiting for developments on the other. Complimenting this approach, Tim chooses projects based on what he finds most interesting. “The more fun it is to work on a project, the more hours you will spend happily and the more effective you will be. It’s worth thinking about, what do you think is useful for the world, but also what will make you happy as a researcher that you can spend a lot of hours on?”

Reflecting on his early PhD years, Tim would tell his younger self to “Try more and think less. That might be counterintuitive, but often, you think you know how things work, and then you try stuff and it’s the complete opposite.”

He warns against overthinking problems instead of testing hypotheses in the lab. “You’ll think for a month about how to solve a problem, then try it, and it doesn’t work.”

The future of proteomics

Looking forward, Tim is excited about two concurrent developments in the field. First is the shift from studying proteins to studying proteoforms – the different versions of a protein based on post-translational modifications (PTMs).

“People thought one gene will encode for one specific protein, but then they found out that one protein can be decorated with different PTMs, each resulting in differently decorated proteins that may have the same amino acid sequence, but they have multiple functions.”

The second development is the move toward understanding glycan structures, not just their compositions. “Initially, people mainly looked at glyco composition… they know the composition, but they don’t know how it’s linked to each other. So they don’t know the 3D compositional structure.”

Tim believes the combination of these approaches – linking glycan structure to specific proteoforms and their functions – will be transformative for medicine development.

“Hopefully soon, we’ll be able to also link it to functional properties of specific glycoproteoforms. Then we’ll be able to link structure to function as one joint story, which is important for the development of new medicine. It’s tiny steps at a time, but all the tiny steps will eventually get us to complete the puzzle. And as the tech keeps developing, those steps will start to get bigger.”

While Tim is optimistic, he recognises the need for extra hands on deck and puts out a rallying cry: “we don’t know everything yet, so there’s still heaps of optimisation that we can do on current mass spec hardware. That’s why I invite all the researchers to enter the glycoproteomics field and join the battle.”

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