The LCGC Blog: Intact Protein Ion Transmission in a Triple-Quadrupole Mass Spectrometer

Jun 28, 2017

I believe that the term “top-down proteomics” holds a particular connotation with respect to the use of ultrahigh-resolution mass spectrometers in people’s minds. And rightfully so. If one is to determine with confidence the sequence and charge state of a particular fragment ion generated in the gas phase, then high mass accuracy is a must. From the discovery side of things, where qualitative analysis is most important, this is not likely to change. However, when you turn to quantitative analysis, where you want to now monitor levels of a particular protein biomarker for the purpose of disease diagnosis, prognosis, or treatment, then invariably bottom-up strategies are the norm. Protein quantitation using top-down strategies, especially on low-resolution triple-quadrupole systems, have been largely ignored, until recently (1,2).

While triple-quadrupole systems are the gold standard for quantitative analysis of small molecules, there are a variety of reasons why you might not initially choose such a system for multiple reaction monitoring (MRM) of intact protein ions. With a low-resolution precursor ion isolation from a multiply charged intact protein ion envelope, multiple adducts, isotopes, and conformations of a protein will be selected. This may lead to quite a heterogeneous mixture of precursor ions that may not all behave identically upon collision induced dissociation. Further, low-energy collisions may not adequately activate the protein for dissociation, since it has many degrees of freedom through which it can redistribute the collisional energy. Finally, if product ions are formed, again one is faced with the use of a low-resolution quadrupole system, so it becomes very difficult to assign the sequence and charge state of any ions generated. With all of that in mind, it is worthy to note that such a strategy does work. If modest collision energies are used, then a series of unique and intense product ions can be formed to carry out MRM-based quantitation. This approach appears to also have the desirable specificity that researchers rely on for small-molecule quantitation.

One issue that we have tried to address more recently is the fact that this top-down approach does not appear to be as sensitive as the bottom-up approach. While we are aware that the efficiency of intact protein ion generation by electrospray seems to be highly dependent on what protein you are monitoring, and that such a concept probably needs more attention and research, we chose to focus more on the ion transmission in a triple-quadrupole instrument, after the ion was formed (3). We believed that aspects of charge transfer, scattering, and mass resolution may be contributing significantly to a loss of signal during MRM.

The details of these experiments can be found in the article, but just briefly some points to highlight include the following items:

  • Intact protein ions seem to be subject to a greater degree of ion scattering compared to small-molecule ions, and such scattering is more pronounced with larger collision gases (for example, Ar and N2, as compared to He). In the transfer of ions through a triple-quadrupole system, you are already throwing away a lot of signal (thankfully, you also throw away a lot of noise too), so minimizing this additional loss for protein ions would seem to be worthwhile.
  • It has been shown previously that multiply charged intact protein ions become more acidic in the gas phase, as their charge density increases (4). Some charge transfer can be observed between multiply charged protein ions and argon as a collision gas. Interestingly, even though nitrogen is listed as being a stronger gas phase base than argon, charge transfer events are largely reduced when nitrogen or helium are used as collision gases. There may be various reasons to explain this, including the limited accuracy of gas phase basicity scales for these weak gas phase bases, or potentially the presence of water in the argon gas; regardless, this was an interesting concept to investigate. Some proteins showed preferential charge transfer to specific charge states (for example, ubiquitin +8 → +5 was more dominant than +8 → +7 or +6), which makes one believe that protein conformation is an important part of this observation.
  • Mass resolution settings on the quadrupoles have a very large influence on ion transmission. We explored whether these effects might be different for protein ions compared to small molecules. Indeed, it seems that some interesting combination of setting the first quadrupole at unit resolution (defined as ±0.35 m/z units on the instrument we were using), while widening the resolution of the third quadrupole (to ±0.7 m/z), created a very large increase in product ion intensity for the intact protein compared to a small-molecule surrogate. This phenomenon is not easy to explain, but may have something to do with maximizing the production and detection of particular product ions from the heterogeneous precursor ion population mentioned previously.
  • Finally, there are some interesting tricks that can be played with summing the signals of multiple MRM events to improve sensitivity. Proteins have multiple multiply charged ion signals from which MRM transitions can be generated. Further, simply summing multiple times a single MRM event (for example, 10 times) seems to provide signal enhancements. Although such capabilities may end up being manufacturer-specific, it seems that in these cases, the signal increases to a greater degree than the noise when these tricks are played. Again, it is not clear whether these same strategies will work equally as well for small molecules or not. I do not believe they have been fully investigated in that regard.

Although some of these concepts might seem rather basic or elementary to a seasoned mass spectrometrist, I believe they do point out some fairly substantial differences in the transmission behavior of large multiply charged proteins ions compared to small-molecule ions in a triple-quadrupole mass spectrometer. As more effort is placed on improving sensitivity for top-down quantitation, these aspects will be more thoroughly investigated. Although many will continue to use established bottom-up approaches, they are inherently more complicated and less suitable for absolute determinations of protein amounts than would be a top-down approach. That said, there are many things that would need to be addressed (for example, availability of standards, internal standardization, and so forth) before top-down quantitation will become very widespread. However, we stand heartened by the comment from one of our reviewers of the most recent article (3). They wrote, “I was left with the overall question: 'I wonder how much better one could do with triple-quads designed for optimal intact protein performance?‘” Indeed, I am very intrigued by this question.



  1. E.H. Wang, P.C. Combe, and K.A. Schug, J. Am. Soc. Mass Spectrom. 27, 886–896 (2016).
  2. K.A. Schug, The LCGC Blog, Aug. 9, 2016.
  3. E.H. Wang, D.K. Appulage, E.A. McAllister, and K.A. Schug, J. Am. Soc. Mass Spectrom. (2017). doi:10.1007/s13361-017-1696-x
  4. J. Sterner, M. Johnston, G. Nicol, and D. Ridge, J. Am. Soc. Mass Spectrom. 10, 483–491 (1999).


Kevin A. Schug is a Full Professor and Shimadzu Distinguished Professor of Analytical Chemistry in the Department of Chemistry & Biochemistry at The University of Texas (UT) at Arlington. He joined the faculty at UT Arlington in 2005 after completing a Ph.D. in Chemistry at Virginia Tech under the direction of Prof. Harold M. McNair and a post-doctoral fellowship at the University of Vienna under Prof. Wolfgang Lindner. Research in the Schug group spans fundamental and applied areas of separation science and mass spectrometry. Schug was named the LCGC Emerging Leader in Chromatography in 2009, and most recently has been named the 2012 American Chemical Society Division of Analytical Chemistry Young Investigator in Separation Science awardee.     

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