Professor D.F. Hunt Relationship with Finnigan Corporation and His Impact on Mass Spectrometer Development

Abstract

Don Hunt’s 50-year collaboration with Finnigan Instruments Corporation has resulted in many innovations in mass spectrometry that range from new instrumentation to new analytical methodologies which in turn enabled new applications. The fruits of this collaboration, directly and indirectly, have had a broad and enduring impact on the instrumentation and practice of modern mass spectrometry. The authors of this monograph were members of the research and development team at Finnigan over time frames that collectively encompass the entirety of the collaboration. This article provides a narrative history and chronology of the Hunt-Finnigan relationship based on their personal recollections as well as the recollections of other participants.

Highlights

• •A partnership that launched Finnigan's successful biological product strategy.
• •A university–industry interaction, fostered by Don, that set a career path for many.
• •Hunt lab prototyped instruments: TSQ, QFTMS, LTQ-FTMS, and technology: ETD, FETD, PTCR.
• •Don, with Finnigan support, taught his “new” peptide sequencing methods to hundreds.
• •The birth of proteomics to rapidly sequence proteins without enzymatic digestion by MS.

In Brief

Don Hunt’s 50-year collaboration with Finnigan/Thermo has resulted in many innovations in mass spectrometry instrumentation and analysis, enabling new applications. It also played a pivotal role in the mass spectrometer product development strategy of Finnigan. This collaboration, directly and indirectly, had a broad and enduring impact on the instrumentation, practice, and practitioners of modern mass spectrometry.

Don Hunt’s 50-year collaboration with Finnigan Instruments Corporation has resulted in many innovations in mass spectrometry (MS) that range from new instrumentation to new analytical methodologies which in turn enabled new applications. The fruits of this collaboration, directly and indirectly, have had a broad and enduring impact on the instrumentation and practice of modern mass spectrometry. (Note: Through the decades the company has been sequentially known as Finnigan Instruments, Finnigan MAT, Thermo Quest, Thermo Finnigan, Thermo Electron and now the Life Sciences Mass Spectrometry business of Thermo Fisher Scientific. For purposes of brevity and to avoid confusion, we will refer to the company as Finnigan.) The authors of this monograph were members of the research and development team at Finnigan over time frames that collectively encompass the entirety of the collaboration. This article provides a narrative history and chronology of the Hunt-Finnigan relationship based on their personal recollections as well as the recollections of other participants.

The year 1967 saw several firsts: Super Bowl I, the premier issue of Rolling Stone magazine, and San Francisco’s “Summer of Love” in the Haight-Ashbury. In addition, Professor Donald F. Hunt received his PhD, and in January, Finnigan Instruments Corporation was founded. The following year, 1968, Finnigan introduced its first analytical quadrupole mass spectrometer instrument, the model 1015 (Fig. 1). That same year Don left his post-doctoral position with Klaus Biemann at MIT and became Assistant Professor of Chemistry at the University of Virginia (UVA). It would be another 6 years before the collaborative relationship between Don and Finnigan would be established, but once established, it would continue for 50 years. Don played a role far beyond the usual academic consulting function. He influenced the instrument development strategy and product development of the company. For this entire period, he consulted for no other mass spectrometer manufacturer and received no personal compensation from Finnigan. The company did support his laboratory monetarily, but more importantly, it provided early access to innovative MS instrumentation, much of which his laboratory had helped develop. The relationship with Don included the usual expectations of a consultant. He communicated new developments from his research early for us to select for commercialization. It also gave the company access to his students, many of whom, as their careers progressed, became users and customers and, in several cases, important collaborators in their own right. Over the years, Finnigan also employed more than a dozen former Hunt students. He also gave seminars to the Finnigan technical and sales staff. However, the relationship was much more than these contributions and perhaps can be appreciated best by understanding some of the history and dynamics of Finnigan.

Fig. 1.

First announcement of 1015 GC/MS, 1968.

Finnigan Instruments Corp. was founded by Robert (Bob) Finnigan and three technical co-founders, William (Bill) Fies, Michael (Mike) Story, and Richard Hein. The founders had no experience in analytical mass spectrometry (MS), gas chromatography (GC), or data systems (DS). Mike and Richard, working for Bob at Electronics Associated Inc. (EAI), had developed a commercially available quadrupole residual gas analyzer (RGA) mass spectrometer for characterization of materials in vacuum systems. The core of the designs for these RGA instruments came from Stanford Research Institute (SRI). Mike and Richard had BS degrees in chemistry. Bill was an entirely self-taught engineer/scientist who specialized in electrical design and developed the electronics for the quadrupole RGA instruments originally built at SRI (1).

Bob, with a PhD in electrical engineering and while working on market research in process control at SRI, had developed a vision that focused on computer-controlled GC/quadrupole MS for the chemist’s bench. Based on their work at EAI and SRI, Mike and Bill were well qualified to build instruments but needed to learn mass spectrometry. Early on, Jonathan Amy, Professor of Chemistry at Purdue University, who had advised on the formation of Finnigan (2), realized this and consented to consult with the company – another relationship that would span 50 years. He became Mike’s mentor, and, with his teaching, Bob and Mike developed an approach to Finnigan’s R&D collaborative interactions with academics and customers (Fig. 2). The relationship with Don Hunt was the most enduring of many such relationships and one of the most impactful on the fortunes of the company and the field of mass spectrometry in general.

Fig. 2.

Relationship between Academia, Customers, and Company.

By the time of Don’s first visit to Finnigan in 1974, the company had regular but low sales from the general analytical laboratory market. Quadrupole mass spectrometers were not well accepted in part due to lower mass resolution compared with sector instruments, which were widely used in academic labs. Quadrupole analyzer-based instruments were not considered “real mass spectrometers,” although they are true mass analyzers. The sector MS instrument companies were happy to exploit these biases. To their advantage, commercial quadrupoles were the only mass analyzers that could be controlled readily by a computer and hence also able to rapidly do what became known as selective ion monitoring (SIM). The only thing lacking in completing their success was instrument control software, i.e., they needed a data system. Finnigan’s first 1015 mass spectrometer (Fig. 3) delivery was to the Stanford University Medical School, where they quickly developed a data system (3) with the hope of doing automatic molecular structure analysis from fragmentation patterns. At Bob’s suggestion, a separate, independent company, System Industries (SI), was formed, and the System 150 Data System (DS) was commercialized by SI people working in the Finnigan lab using the Stanford design (4). Submission of the company’s new hybrid product, the 1015 GC/MS/DS, to the newly formed U.S. Environmental Protection Agency testing facility was so successful that the subsequent sales kept the undercapitalized company from closing. The primary business of the company became building GC-MS instruments for priority pollutant analysis (e.g., the EPA Clean Water Act of 1974). It was the first “killer application” for mass spectrometry.

Fig. 3.

Finnigan 1015 with prototype data system at Stanford University.

Chemical Ionization Quadrupole MS

To increase capability for the analytical laboratory market, in 1970 Finnigan introduced a quadrupole instrument with a chemical ionization (CI) source developed by Mike in cooperation with Marvin Vestal, then with Scientific Research Instruments. Don, as part of his tenure-associate professorship award, was funded for the GC/CI/QMS/DS system and came to the company to give a seminar and participate in a demonstration of the instrument. Don already had a Vestal-designed CI source on a sector instrument (AEI MS902) and so had insight into the increased analytical capabilities of the computerized quadrupole GC/MS/DS system. Although his funding was insufficient to cover the cost of a full instrument, Mike and Bob were impressed enough with Don’s enthusiasm and his knowledge of using chemistry with MS to solve problems that they stripped out the GC and DS to reduce Finnigan’s loss and shipped him an instrument priced at $40K (below cost). The first night of Don’s visit, Mike took him to a “sit on the floor” Japanese dinner, his first. That almost scuttled the nascent relationship; however, the sake was good, and a strong bond was forged between Don and Finnigan, which began the career-long connection that would benefit both many times over.

Many of the cooperative projects that Finnigan had with Don are well enumerated by Yates et al.(5) and augmented in this issue (6, 7, 8). These were projects mostly initiated by Don, but each project added analytical capability, and more importantly, influenced the direction of Finnigan’s longer-term instrument development strategy ever further towards life science applications.

In the early days of the company and into the early 1980s, Finnigan’s R&D funding (10–15% of sales revenue) went mostly to environmental instrument development and much less to the development of those for the life sciences. There was little application overlap between the two technologies, except in data systems. As the environmental market instruments' analytical requirements narrowed, Finnigan’s environmental offerings became less competitive than other manufacturers, most notably, offerings from Hewlett-Packard Co (later Agilent). Finnigan’s strength was in developing instruments that were innovative, offering high performance and novel capabilities, rather than the refinement required to develop lower-cost instruments with robust but basic analytical functionality and performance that best served the environmental market.

Don’s suggestions and those of other academic consultants supported the internal desires of the “S guys” to increase internal funds for the development of instruments with the capability to serve the emerging Life Sciences markets. Who were the “S guys”? Finnigan had a remarkable number of scientists whose last names began with S: George Stafford, John Syka, Steve Sokolow, Urs Steiner, Alan Schoen, Jae Schwartz, Mike Senko, and Mike Story (the original). Because he facilitated all the Finnigan/Hunt lab interactions, Jeff Shabanowitz has been an honorary S guy. Jeff came to UVA in 1976, earned his PhD with Don in 1983, and has managed the Hunt lab to the present day. In recognition of his achievements in that position, in 2019 Jeff was awarded the first American Society of Mass Spectrometry (ASMS) Al Yergey MS Scientist Award that acknowledges “unsung heroes” in the field. He received this award for his 45 years of dedicated support in the Hunt Laboratory, managing the diverse research programs, mentoring graduate students, and providing strong collaborative engagement with Finnigan Corporation to enhance our special relationship.

In 1976, 2 years after Don’s first visit, he called Mike to encourage Finnigan to hire his graduate student, George Stafford. As part of his graduate work, George developed negative ion capability on that first CI instrument (Finnigan 3200 QMS, upper mass range of 800 m/z). After completing his degree at UVA, George joined Finnigan in October 1976 and was initially tasked with commercializing the Pulsed Positive Negative Ion Chemical Ionization (PPNICI) accessory for quadrupole mass spectrometers. The PPNICI product was successfully introduced in 1978 and, at the time, gave Finnigan a strong competitive advantage in the GC/MS marketplace. Mike remembers George commenting in those early days on the complexity of manufacturing multiple products as opposed to just one. This highlights the intricacies and challenges that come with scaling up for the commercial market.

For the negative ion CI research at UVA in the mid-1970s and its subsequent commercialization by Finnigan, Don received the 1994 ASMS John B. Fenn Award for a Distinguished Contribution in Mass Spectrometry. The ASMS award acknowledged pioneering work in the development and application of negative ion CI mass spectrometry. It also recognized that negative ion CI yielded significantly increased ion current and unique structural information. The write-up for the award stated that “negative CI is a standard feature on commercial analytical mass spectrometers and a methodology that is widely used in the pharmaceutical industry, analytical toxicology, and environmental analyses for the detection and quantitation of trace levels of a wide variety of organic molecules including drugs, drug metabolites, and environmental contaminants.”

Triple Quadrupole MS

Graham Cooks at Purdue University, also a long-time Finnigan consultant/collaborator, had alerted Mike that Rick Yost and Chris Enke would present a paper on their triple-stage quadrupole development at the ASMS meeting in 1978 (9). After attending the presentation, Don approached Mike to say he wanted to build a triple quadrupole mass spectrometer. In the summer of 1978, after cobbling together parts from the 3 existing single GC-MS systems in the Hunt lab at the time, Don and Jeff demonstrated that the technology would work for analytical purposes (6). Don convinced Mike that Finnigan should make it a product and guaranteed five immediate sales. At the time, it took 2 to 3 years to develop and bring to market a new instrument. Finnigan had engaged a consulting firm to “systematize” manufacturing methods a year before, which had increased the development time (more paper and procedures but hopefully higher quality and lower manufacturing cost). Mike, knowing that to interfere with the current product development cycle was impossible, prepared a first-of-its-kind (for Finnigan) “skunk works” proposal guaranteeing sales with the usual gross margin but with a 10–12-month development cycle. The instruments were to be designed and constructed (somewhat like experimental race cars) by a small team within the research group managed by Mike. Mike presented the proposal to TZ Chu (then CEO of Finnigan), who was known to resist anything that would disrupt the new manufacturing scheme. With Bob’s and Don’s support, however, TZ accepted the proposal with one correction on pricing. He doubled the profit margin, saying, “For once we will price according to value, not costs”. The skunk works project to develop the TSQ was unique, never successfully repeated at Finnigan, and was only approved because of Don’s relationship and history with Bob and TZ. Figure 4 shows the diagram of the triple quadrupole instrument constructed by Jeff in the Hunt lab using components from 3 GC/MS instruments. This research and a description of the UVA instrument was first presented at the 1979 Seattle ASMS Conference and later published (10, 11).

Fig. 4.

Diagram of the triple quadrupole instrument built in the Hunt lab by Jeff Shabanowitz.

Data System

Mention has been made of the critical role of mass spectrometer data systems. The Hunt lab never developed a data system (DS) of its own, but in 1976 Don recommended that Bob consider buying a startup mass spectrometer data system company located in Berkeley, CA named INCOS. Finnigan had acquired a small company earlier that had a push button x-ray fluorescence data system that was easily converted to mass spectrometry, and it had become a standard Finnigan offering. However, the DS was not well accepted by laboratory scientists as its operation was inflexible and opaque. The INCOS data system was the opposite: full accessible control of multiple quadrupole mass analyzers and for its time both a convenient keyboard command interface and high-quality graphical representation of MS data. Its ability to handle high data acquisition rates and manage foreground/background processing was needed for the more complex automated scanning required for the TSQ. The company was purchased and after a major revision by Joel Karnofsky, its original developer, the INCOS MS data system became the standard of the industry. This system, which had been suggested by Don, was at the core of all Finnigan products into the mid-1980s and remained so for the products intended for use in environmental markets until it was retired in the mid-1990s. When the scanning, data analysis, and information output of the INCOS system were combined with automated ion source tuning, the GC/MS/DS met the requirements of the massive analysis of millions of environmental samples.

The instrument control functionality of the INCOS data system was key to quickly developing a fully computerized triple stage quadrupole (TSQ) system in 1980. INCOS was originally designed to control two mass analyzers (for its original market of a double-focusing sector MS). This multiple-spectrometer control enabled data-dependent MS/MS instrument operation. This allowed, during a chromatographic run, the selection of the major peaks of eluting analytes in the first quadrupole that, with a change in instrument configuration, could be fragmented in the middle quadrupole collision cell and their constituent parts analyzed in the third quadrupole. This could be accomplished automatically and, although not highlighted at the time, suggested a potential path to automated protein sequencing. A TSQ production prototype was designed and built, INCOS software modifications were made, documentation was written, and the system was tested all within 12 months by Urs Steiner, Steve Sokolow, Charles Boitnott, and John Slayback. The TSQ development team members were selected/recruited from different departments of the company by Mike, who was in charge of the project. Delivery started in 1981 (the first commercial TSQ), at least 2 years ahead of the usual product development cycle. With the combined efforts of Don, Bob, Mike, and the Finnigan sales force, more than 10 systems were sold in the first year. One of the first to be delivered was to the Hunt lab, with payment completed later after successful grant proposals provided funding. This funding and purchasing pattern was typical between Don and Finnigan and was repeated for the acquisition and purchase of many instruments through the years including next generation TSQ instruments (TSQ 70 and TSQ 700), and various versions of LCQs, LTQs, an LTQ FT, and an LTQ Orbitrap.

Teaching the World to Sequence Peptides

In the early 1980s, with the development of ionization methods such as Fast Atom Bombardment (FAB), Don fully committed the work of his laboratory to the development of MS-based methods for peptide analysis. By the second half of the 1980s, the Hunt lab had developed tandem mass spectrometry methods using triple quadrupole instruments for peptide sequencing and had demonstrated results that were superior to those obtained using the vastly more complicated and expensive four-sector tandem mass spectrometers. The four-sector tandem MS community adamantly argued that these were the only instruments suitable for that application. There was skepticism about the ability of other labs to implement Don’s methods. To support Don’s approach of peptide sequencing using low-resolution quadrupole MS (and sell more TSQs), Finnigan supported and promoted a series of classes taught by Don and Jeff at the UVA from 1989 to 1995. They were 4 days long and included laboratory demonstrations and demanding homework assignments involving manual interpretation of triple quadruple MS/MS spectra. With ∼20 attendees per class, Don, along with Jeff and students, eventually trained more than 400 scientists from around the world (including several Finnigan employees) in the Hunt lab methods for de novo sequencing of peptides using triple quadrupole MS data.

RF Ion Traps

By 1983, George Stafford had been working as a research scientist and instrument developer at Finnigan for over 6 years. His work included further development of chemical ionization, negative ion detectors, and an ion source design that resulted in the development of the interchangeable ion volume EI/CI source for the Finnigan 4000 series and triple quadrupole series instruments. He also invented a new way of scanning quadrupole RF ion trap analyzers (Mass Selective Instability) that enabled combining the ion trap with other mass analyzers. By mid-1983, George was the lead scientist driving a crash program to develop a product based on his invention, which would serve as the basis of a m/z scanning GC detector (ITD 700). As is typical for the development of any new technology, the development team encountered many unanticipated problems, and the project was behind schedule.

In 1983, Don offered George a 9-month (Sept 1983 to May 1984) graduate-level teaching position at UVA and a collaborative research assignment to work with Jeff Shabanowitz and Don’s graduate students. George discussed this opportunity with Mike, and they decided George should accept the offer because it would be beneficial to George and Finnigan, and it would also nurture the relationship with Don and his group at UVA. While in Charlottesville and on leave from Finnigan, George taught graduate courses on analytical chemistry and instrumentation technologies. His collaborative research with Finnigan at UVA during this period focused on continued investigation of quadrupole RF ion trap mass resolution improvements based on He, H_2, and other gas additions to the ion trapping volume using an ion trap instrument provided by Finnigan. His “sabbatical” at UVA removed George from the daily pressures of the ion trap development project and allowed him to think more deeply about technical problems besetting the project. George appreciated and was impressed by Finnigan's full support and willingness to accommodate his temporary teaching and collaborative research assignment at UVA. This level of cooperation and support between an instrument manufacturer and a university is possible only when there is complete trust and shared goals between the partners.

Early in 1984, the ion trap MS development project had reached a crisis point regarding the ion trap performance. An emergency meeting of senior management, project team leadership, and Finnigan’s principal academic consultants and collaborators—the corporation’s brain trust—was organized to evaluate data and decide what to do. Cancellation of the product introduction was a highly possible outcome. The meeting was held on Super Bowl Sunday, and attendees included John F. J. Todd from the University of Kent (Canterbury, UK), Graham Cooks, and Jon Amy, who was on sabbatical from Purdue and running Finnigan’s R&D group while Mike was in Japan establishing the company’s office there. George and Don traveled to San Jose for the meeting.

The most perplexing and worrisome of the ion trap technical problems enumerated at the meeting was that the relative abundances of the molecular ion ¹³C isotopic peaks to that of the ¹²C peaks varied as the sample eluted into the ion trap. At maximum abundance (the top of the GC peak) the isotope peaks increased in relative abundance quite markedly. After reviewing the data, Don provided the explanation: at the maximum sample partial pressure, analyte ions (created by electron ionization) and analyte neutrals were undergoing ion-molecule reactions forming protonated molecules that were isobaric with the ¹³C isotopic peaks. Don argued unpersuasively that this was a positive feature as it indicated what peaks were from molecular ions. The problem could be resolved by limiting the amount of sample delivered to the ion trap, however, there was concern that the instrument would not be sensitive enough to produce interpretable spectra from less sample. George remained in San Jose for a few days after the meeting to prove that he could acquire high quality spectra using lower sample amounts. In other words, he was able to show that the ion trap analyzer was extremely sensitive. He also solved a second seemingly unsolvable problem. He eliminated the need to adjust the trapping RF voltage level to achieve acceptable m/z resolution across the full m/z range of the instrument—“the tunable mass range problem.” After these accomplishments, he returned to Charlottesville. Thus, over a few days in late January 1984, Don and George saved the project to commercialize the RF quadrupole ion trap and perhaps prevented the company from abandoning RF ion trap technology (12).

Quadrupole Fourier Transform MS

It was an exciting time in the Hunt lab after Michael (Mickey) Barber, in 1981, introduced Fast Atom Bombardment (FAB) (13), which provided an efficient way to produce gas-phase peptide and protein ions, and the TSQ was being put through its paces. Don was in the process of transitioning the focus of his research from the application of negative chemical ionization MS and tandem mass spectrometry for the analysis of “small molecules” (in the lexicon of 2024) to the use of mass spectrometry to characterize peptides and proteins (14). The development of FAB ionization might enable this, but it has not been practically realized. The quadrupole-based instrument lacked the high resolution required for high-mass ions of biomolecules. While Don was on sabbatical (1981–1982) in Howard Morris’ lab at Imperial College, he and Jeff discussed the next generation of instrumentation for biological molecules. They envisioned externally injecting ions into a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. Don knew the potential of such a high-resolution tandem quadrupole-FT-ICR MS instrument that did not require the high accelerating potentials of sector instruments. Further, the FT-ICR offered the high m/z range needed for analysis of the singly protonated peptide/small protein ions. Don was intrigued by the notion that m/z analysis in the FT-ICR was theoretically non-destructive; something that was much discussed at the time in the ICR community, though, in the end, never implemented in an analytically practical manner. Potentially, a single accumulated batch of ions could be re-analyzed multiple times or subjected to both an MS and a data-dependent MS/MS experiment. Don knew this could be very useful in the analysis of biological samples where sample amounts would be low. After returning from his sabbatical, Don reached out to Mike about developing the new instrument.

Although Don did not know it, Finnigan was already considering the development of such an instrument. Robert (Bob) McIver, a professor of chemistry at U.C. Irvine, had approached Finnigan with a similar idea. A state-of-the-art FT-ICR instrument in 1982 required the ion source to be near the ICR cell and hence inside the bore of the superconducting magnet. Bob approached Mike with an idea to inject ions from a source distantly located from the ICR cell and outside of the bore of the magnet. His proposal was to use a long RF quadrupole ion guide to transmit the ions axially into the magnetic field. The strong radial confinement provided by the RF quadrupole field would deliver ions efficiently to the ICR cell and thereby overcome the repulsive “magnetic mirror effect” induced by the solenoidal magnet fringe fields. This would increase the analytical utility of FT-ICR instruments as it would allow the use of high gas load sample inlets and ionization sources such as GC, chemical ionization, and the recently developed FAB technique, the last of which enabled the production of gas-phase ions of staggeringly large (for the time), non-volatile biological molecules like peptides. The high resolution and accurate m/z measurement capabilities that FT-ICR promised were well beyond what the company’s (or any other manufacturer’s) magnetic sector instruments provided.

To pursue this vision, a new Finnigan-university technology development project was initiated to develop a quadrupole-FT-ICR MS instrument. It was a three-way collaboration between Bob McIver, Richard (Rick) Hunter, and their small startup company (IonSpec), Finnigan, and Don, with instrument development continuing separately in the Hunt and IonSpec laboratories. It was a highly speculative and risky endeavor, and Mike and Don sought academic and industry partners who would commit to ordering the instrument when it became commercially available and potentially provide upfront money that would help fund instrument development. This was the same approach used just 3 years earlier by Bob Finnigan and Mike to develop the company’s first triple quadrupole MS instrument, the TSQ. Finnigan purchased 7-T magnets for the Hunt and IonSpec laboratories. An FT-ICR instrument using a then state-of-the-art 7-T superconducting magnet would provide isotopic resolution of a singly charged 5000-Da small protein ion with a one-second “scan” (transient) time. In 1983, this was a mighty impressive prospect.

In early February 1983, John Syka, a young research engineer working for Mike, and Michael Labunski, a mechanical engineer, cobbled together a vacuum system and analyzer assembly, and John delivered it to the IonSpec laboratory in Tustin, CA. Over a long weekend, Bob McIver, Rick, and John assembled the instrument and demonstrated that ions could be transmitted efficiently into the bore of the solenoid by a long quadrupole ion guide. While this setup was appropriate for the proof-of-concept experiment, as also demonstrated by Jeff’s system being built concurrently in the Hunt lab, it was not a proper design for an analytically useful instrument. In particular, an improved vacuum system was needed, and Jeff designed one based on cryopumps. John fabricated some of the parts at Finnigan, and shipped them to Jeff, who constructed the second instrument in Charlottesville in early 1985. The ICR cell controller, signal acquisition system, and spectral display system came from IonSpec (Rick Hunter’s design). In mid-1985, John made his first visit to the Hunt laboratory. He spent a few days modifying the RF ion guide electronics in the hopes of improving the effective m/z range of the instrument. This project was the start of a 40-plus-year collaborative relationship between Jeff and John that continues to this day.

The new tandem quadrupole Fourier transform mass spectrometer (QFTMS) (Fig. 5) enabled ions produced in a high-pressure ion source to be transmitted efficiently through the superconducting fringing field (including a 1-m long Q2) and trapped in the ICR cell allowing acquisition of high resolution (>10K) mass spectra of large (∼1-2 KDa) peptides. The data provided “considerable encouragement in our effort to develop a new approach to biopolymer sequencing…” (15, 16).

Fig. 5.

Diagram of the Quadrupole Fourier Transform mass spectrometer.

Electrospray Ionization

Electrospray ionization (ESI), introduced by John Fenn and co-workers in 1988 (17), enabled ionization of biomolecules and, with multiple charging, made the mass range of quadrupoles (about 2000 m/z) ideal. Don argued for a change of focus to ESI but was met initially by resistance at Finnigan. However, by the mid to late 1980s, it was clear to Don and a few others that the future mass spectrometry market for Finnigan would be in the life sciences field. The influence of Don and others prompted Mike to propose to company leadership that Finnigan develop an analytical biochemistry focus by hiring a respected scientist in the field. In consultation with many people, including Don, a list was drawn up, and Mike visited Dr Ian Jardine of the Mayo Clinic, who was first on the list. It was winter in Minneapolis, and he and especially his wife was an easy sell for moving to California. When he joined Finnigan in June of 1988, plotting a future strategy for the company had become more complicated than it had been a decade earlier when environmental applications were king. The new environment required a deeper understanding of biological applications. With Ian, Finnigan now had an in-house expert to help prioritize application challenges that could be addressed by the company.

For Finnigan, the adoption of ESI was complicated because the intellectual property rights for the glass capillary-based inlet developed by the Fenn and co-workers were controlled by Analytica of Branford. Finnigan was waiting for Analytica of Branford to develop an atmospheric pressure interface for Finnigan’s triple quadrupole instruments. Don was certain that his triple quadrupole-based methods for peptide analysis would be applicable for the multiply-charged peptide ions generated by ESI and that ESI was the future for MS-based peptide sequencing. He wanted to obtain the first of these interfaces when they were delivered to Finnigan. Ian Jardine wanted the Finnigan R&D group to evaluate the interface. Mike convinced Ian that, in the long run, a relatively small delay to the Finnigan R&D team would be more than compensated for by the advantage gained when Don presented and published results obtained using Finnigan triple quadrupole instruments with the new interface. Don obtained the Analytica interface, and events evolved as Mike predicted. Don and Ian were the individuals most responsible for redirecting Finnigan’s product development strategy toward building instruments for biological applications.

Another aspect of the Finnigan/Hunt relationship was the interaction with other company consultants. During Nathan Yates’ second year of graduate study research with Prof. Richard Yost, University of Florida (also a long-time Finnigan consultant/collaborator), he spent 6 months on a research project at Finnigan under George’s direction, working with the ion trap development team. He focused on the development of software, Ion Catcher MS (ICMS), that automated the process of setting up ion trap MS/MS configurations based on previous scans. After graduation, he went on to a post-doc at UVA with Don and continued his ICMS development and further advanced MS/MS methods. This work not only supported the research objectives of the Hunt lab but also provided a preview of ion trap MS-based analytical techniques for life science applications.

Don’s continued collaboration with Mike, George, and John benefited the Hunt lab and Finnigan. As with Don, Finnigan shifted its focus to the analysis of biological samples using the new instruments, which opened new markets and new approaches to biochemical research. Dozens of papers were authored by Don, many with his Finnigan collaborators. Don was awarded an NIH grant (1/12/1987), Protein Sequencing by Tandem Mass Spectrometry, which sustained his lab for the next several decades. This opened the door to the study of post-translational modifications, protein-protein interactions, and further innovations in instrumentation.

Quadrupole Linear Ion Trap-FT-ICR

In late 1996, John, who Mike had brought into the Finnigan research group as part of the student summer hiring program in 1978, decided that it was time for him to follow the path of other Finnigan colleagues, and go back to school to pursue a doctorate. John was trained as a mechanical and an electrical engineer and was very mathematics and physics oriented. The UVA School of Engineering had a PhD granting program in Engineering Physics that suited John’s education and expertise. To benefit Finnigan, John determined that if he worked in Don’s laboratory on biological mass spectrometry, he would be exposed to challenges for which he could help create MS instrument solutions. George Stafford, who was John’s boss, was very supportive. Because John had a track record for innovation, Finnigan elected to pay John’s graduate school tuition and maintain him as a company employee (at significantly reduced salary) so that the company would have some claim to any intellectual property resulting from his work. This arrangement would allow John to access Finnigan proprietary and confidential tools and technical documentation and with an entirely different level of material support than most graduate students. John could obtain his PhD and facilitate the collaboration between Don and Finnigan. An agreement was reached between UVA and Finnigan stipulating that any new technology resulting from John’s ideas and efforts would belong to Finnigan, whereas ideas and efforts from the Hunt lab would belong to UVA. Finnigan was given the right of first refusal to license the work that was patented by UVA. In April 1997, John moved with his family to Charlottesville, and his arrival began yet another productive chapter in the Hunt-Finnigan relationship.

On John’s first visit to the Hunt Lab after moving to Charlottesville, he met with Jarrod Marto, who was a post-doc in Don’s lab, having received his PhD from Alan Marshall’s lab at the Ohio State University. Jarrod was working on changes to the QFTMS instrument that Jeff and John had assembled over a decade earlier. Jarrod had converted the instrument to a highly sensitive, high-m/z resolution LC/MS by adapting an ESI source from one of Finnigan’s TSQ instruments. This resonated with John, as he had been thinking about a radial ejection RF linear ion trap analyzer-based MS and had submitted an internal patent disclosure for the idea in 1987. He wanted to pursue it as part of his dissertation work. It occurred to John: Why not put a linear ion trap analyzer in front of the ICR? For MSⁿ experiments, it could perform all the m/z selection and ion activation, and then the product ions could be transferred to the ICR cell for high-resolution m/z analysis. John and Jarrod talked about it over lunch. The linear ion trap analyzer with its ion detector would have the capability to regulate the number of ions (charges) it accumulated (Finnigan term is automated gain control, AGC). The linear trap analyzer could regulate the number of charges delivered to the ICR cell. This would be advantageous, as the variance in the space charge in an ICR was the primary limitation in FTMS m/z accuracy. AGC would enable high m/z resolution and high m/z accuracy without using internal m/z standards.

By the end of that lunch, Jarrod was fully on board with the concept; however, such an instrument-building project would require a lot of resources and support from both sides of the collaboration. At first, Don was not receptive to the idea because he had his sights set on a second ESI-enabled quadrupole FT-ICR instrument; however, eventually, he supported the plan. On Finnigan’s side of the collaboration, Rienhold Pesch (Finnigan, Bremen, Germany), made multiple visits to the Hunt lab to work with Jarrod to assess the capabilities of the quadrupole FT-ICR instrument. In July 1999, it was decided that a team led by Stevan Horning would develop an FT-ICR analyzer that could accept ions from a quadrupole linear ion trap that was under development in San Jose. Finnigan committed to providing material support to enable Jarrod and John to build a proof-of-concept hybrid quadrupole linear trap FT-ICR instrument in the Hunt lab.

Jarrod and John designed and constructed the hybrid linear ion trap instrument and were able to obtain spectra prior to the ASMS conference in 2001 where John gave a presentation (18) (Fig. 6). Finnigan introduced the LTQ-FT-ICR at the 2003 ASMS conference. In November of that year, one of the first LTQ-FT-ICR instruments was installed in the Hunt lab, displacing the proof-of-concept instrument (See the J.R. Chapman article in this issue), and the first paper describing the instrument was published in 2004 (19). The performance of the LTQ-FT-ICR exceeded what Jarrod and John had envisioned in April 1997. It was the first true high resolution accurate mass instrument. Two years later in 2005, the company introduced the LTQ-Orbitrap, and within a few years the LTQ-FT-ICR product line was discontinued.

Fig. 6.

Prototype (left) and schematic (right) of the LTQ-FT-ICR in the Hunt lab.

Electron Transfer Dissociation

No sooner was the prototype LTQ-FT-ICR operational and generating data when Don, as always, moved on to the next challenge. The LTQ-FT-ICR allowed rapid peptide sequencing with high resolution and sensitivity. The Hunt lab now focuses on peptide/protein post-translational modifications (PTMs) and showed that phosphorylation and glycosylation are important not only in proteomics in general but also in immunology and glycobiology (20, 21, 22). Collision-based MS/MS methods were limited in the capability to localize PTM sites due to fragmentation-induced loss of labile amino acid modification moieties. McLafferty and Zubarev demonstrated that electron capture dissociation (ECD) would significantly advance the sequencing of PTMs (23). Don was eager to try ECD with the LTQ-FT-ICR; however, at the time, each ECD MS/MS spectrum required 1 to 2 min of acquisition time due to the low abundance of thermal electrons. How to overcome this? Don, John, and Jarrod began discussing ideas for electron transfer dissociation (ETD). The proposal was to generate sufficient thermal electrons from negatively charged organic molecular ions produced using chemical ionization techniques developed in the Hunt lab 30 years prior (see J.J. Coon et al. in this issue). In 2002, Josh Coon joined the lab as a post-doctoral student fresh out of the University of Florida with an NIH Fellowship for research on atmospheric MALDI. Don approached Josh about working on the ETD project with John, but Josh was hesitant because he had a fellowship to study something else. Don, in his usual confident and unwavering manner, dismissed Josh’s concerns and the rest, as they say, is history.

Josh and John got to work implementing ETD by generating reagent ions in a back-end ion source of an LTQ instrument. They worked on adding the new CI source and modifying the instrument control code. The development of ETD also required finding one or more chemicals that would yield negatively charged reagent ions that would transfer sufficient thermal electrons to the peptide to result in fragmentation. John and Josh experimented with many potential ETD reagents (see J.J. Coon et al. in this issue). Most reagent candidates produced a mix of ETD fragmentation and ion-ion proton transfer (IIPT), first described by Stephenson and McLuckey in 1996 (24). Josh and John settled on using anthracene for efficient production of ETD reagent ions (25), but it was clear that IIPT (later termed Proton Transfer Charge Reduction or PTCR by Finnigan) would also prove useful in proteomics analysis (26). By 2004, ETD was working beautifully on a prototype LTQ in the Hunt lab, and it was introduced by Thermo as a product in 2006.

For Don, the next logical step was to implement ETD on a high-resolution instrument. Adding ETD to the LTQ_FT-ICR would allow for the resolution of more highly-charged precursor ions, which in turn would play to the strength of ETD. Don was interested in exploring larger peptide and intact protein analysis and identification of multiple PTMs. To enable ETD on the LTQ-FT-ICR would require moving the ETD ion source from the back end to the front end of the instrument. This was challenging as it would require adding an ETD negative reagent ion source near the ESI positive ion source on the front end of the instrument. The first step would be to demonstrate that negative ions could be generated on the front end, trapped and detected. Jeff accomplished this using an older LCQ instrument in the Hunt lab so that he would not disrupt ongoing work (Fig. 7 left panel).

Fig. 7.

Left, Proof of principle using a discharge to make reagent ions and inject into an LCQ. Right, testing prototypes for front end CI/discharge source designs on an LTQ.

After Jeff obtained proof-of-principle results, Don wanted to design a new source for the front end of an LTQ combined with the higher resolution FT and Orbitrap instrumentation. Jeff and Phil Compton, a new graduate student in the Hunt lab, started working on the front-end source for the LTQ in 2006 with the help of Lee Early, Chris Mullen, and Jae Schwartz at Finnigan. This task required both a discharge source for ETD negative reagent ions and a source for positive ions, so they worked on creating a pulsing source by rapidly changing lens polarity. The new source was added to the outside of the LTQ (Fig. 7, right panel). In 2011, the proof of concept had been established, and Finnigan supplied an LTQ Orbitrap Velos instrument “on loan” to the Hunt lab for continued collaboration on front-end ETD (FETD). In the Hunt lab, this instrument was often in various states of modification and testing. Jeff and Phil worked closely with Lee, Chris, Jae, and others to move the source to the inside of the instrument as part of the ESI source (27, 28). Thermo introduced FETD to the mass spectrometry market in 2013, and the first commercially available FETD Tribrid mass spectrometer was the Orbitrap Fusion instrument, which arrived in the Hunt Lab in 2014.

Work on the refinement and application of ETD continued in the Hunt lab, and weekly meetings with Finnigan stimulated new ideas and experiments on both sides. Don continued to emphasize the need to move away from small tryptic peptides to intact protein analysis, and the collaboration turned to methods to meet this goal. There were (and still are) many challenges to overcome. Fragmentation of large peptides and intact proteins can yield overlapping m/z ions with low S/N, resulting in highly complex spectra that are difficult to interpret. In addition, highly charged ETD fragment ions can continue to react with the reagent ion, causing further fragmentation. Experiments showed that the products of sequential ETD reactions could be stored in the C-trap, resulting in a linear increase in the number of ions analyzed, which consequently increased the signal over noise. Work also continued on PTCR, which could be used to abstract protons from ETD fragment ions, thus decreasing fragment ion charge states and spreading the signal over a wider mass range. Controlled PTCR experiments produced spectra that were far less complex and easier to interpret without a loss of informative fragment ions. McLuckey (29) first described parallel ion parking (PIP) as a method to slow the reactivity of ETD fragment ions. John and Chris Mullen worked with several students in the Hunt lab to successfully implement PIP on the Velos instrument (30, 31). This further reduced the complexity of ETD spectra of intact proteins and made the practical use of ETD for top-down analysis more feasible. In 2019, PTCR became available on Thermo Orbitrap Eclipse Tribrid MS instruments. The technology was also made available to the Florida State University National High Magnetic Field Laboratory for use on their 21-T FT instrument (32, 33).

Concluding Thoughts

The 50-year relationship between Don and Finnigan/Thermo was far reaching (and lasted through at least five name changes for Finnigan along the way). During that time, numerous advancements and innovative changes have occurred in mass spectrometry instrumentation and application, many of which were directly inspired by this productive collaboration. There were many contributors over the years from Finnigan and the Hunt lab, too many to mention here and to whom we all owe a big thank you. All sides benefited, and personal friendships were built. New instrumentation (TSQ, QFTMS, LTQ-FTMS), new methodologies (ETD, FETD, PTCR) and new applications in peptide sequencing contributed (and continue to contribute) to revolutionary changes in the study of biochemistry—the birth of proteomics, the identification of PTMs, the study of immunologically relevant peptides, our understanding of histone function and the ability to sequence proteins without enzymatic digestion. With the support of Finnigan, Don taught his peptide sequencing methodologies (both CID and ETD) to hundreds of scientists and students (and continues to do so; see L.C. Anderson et al. in this issue). There is no doubt that the fruits of the collaboration will continue to have an impact on future generations of scientists and may well serve as an example for future collaborative efforts between academia and the private sector. Many people were responsible for the success of Finnigan, but there is no doubt that Don, Jeff and their students were instrumental, pun intended.

Conflict of Interest

The authors declare that they have no conflicts of interest with the contents of this article.