In this article, we examine how tandem and tandem hybrid mass spectrometry has opened up new frontiers already. We go further
and examine how lesser-known experiments are breaking new ground, with alternative fragmentation techniques, as well as the
addition of extra levels of orthogonality by parallel separations techniques.
Today, in the biopharmaceutical industry mass spectrometry (MS) is a critically useful and efficient tool for routine and
investigational analysis in therapeutic discovery, development, and production. Almost every analytical department now routinely
uses MS at some stage in the process of therapeutic development.
However, there is one intriguing aspect that is somewhat surprising given the prevalence of MS; that is, a monolithic view
held by some whereby all mass spectrometers or all techniques are lumped into a broad category labeled "MS." This is all the
more surprising given that the experiments performed are enormously varied. It is the view of these authors that such convenient
shorthand results from a predominance of a small number of MS experiment types adopted by the industry. Although many may
know that alternative experiments exist, few have the time to explore them and many may be unaware of the extreme utility
of these experiments for greater efficiency and information, with little time penalty or method development. In this article,
we touch on how tandem mass spectrometry (MS-MS) has developed and how alternative uses of it may better inform the industry
and speed up therapeutic design and development, with particular reference to the biopharmaceutical area.
A Brief View of History
The development of MS-MS has not been seen as obvious, and has relied partly on fortuitous results and typical scientific
curiosity about fundamental gas phase reactions (1,2). In the 1970s the use of MS-MS was extremely informative about the behavior
of ions in the gas phase and their dissociation, although it remained highly academic (3,4). In experiments that often used
enormous magnetic sector instruments, advanced research was still looking intently at what was later termed "fundamentals,"
reflecting how the field was aiming to understand the very mechanisms of what was occurring (5). In fact, MS-MS research had
been a steady thread of activity right from the very start of mass spectrometry, beginning more than a century ago (1). However,
the 1970s saw the massive rise of a plethora of instrument types, including some ambitious multiple-sector instrumentation.
One type was the tandem quadrupole, which opened up what has arguably been the most commercially successful type of mass spectrometer
ever invented, and which still dominates the market today (8). In common terminology, this has become known as a triple quadrupole, on the basis that the middle quadrupole segment was the collision cell, although this mechanism has long been superseded.
Figure 1: Graph showing the estimated relative adoption rates of MS-based detection versus optical detection for the biopharmaceutical
market (2014–2018). Both techniques grow above 5% per annum, but MS-based techniques accelerate as more biotherapeutics reach
the market and pipeline. (Data sources: various, including FiercePharma, PhRMA reports, public company reports; collated by
But here too lies one of the continuing puzzles for many people in the field: Why has the variety of experiment types not
been used more widely? The most predominant MS-MS experiment remains that used for quantification of analytes: multiple reaction monitoring (MRM), whereby a precursor is selected, and a small subset of the fragments are subsequently monitored to determine very precisely
how much of the analyte is present — mostly with reference to isotopically labeled standard analog species. However, almost
all tandem mass spectrometers have the inherent capability of looking "backward" by using the fragment ion species to reconstruct
what the precursor molecule was like. This has been extensively explored in the metabolite identification world — for example,
where predictable biotransformations can be mapped by integrating the MS and MS-MS information with informatics packages (10).
Additionally, it is also possible to look backward and mark out the parts of a molecule that are not present because they
didn't ionize or were broken into pieces that are not recognizable. Examples of this are "constant neutral loss" experiments,
or "parent–precursor ion scans" (13). It is all the more surprising that these types of experiments are not performed more
frequently because they can be done almost simultaneously in certain types of mass spectrometers (for example, tandem quadrupoles
and quadrupole time-of-flight [QTOF] systems).
Figure 2: (a) An example of an automatically assigned peptide map by LC–MS-MS where each of the peaks is labeled by software.
The panels at the bottom indicate the orthogonal evidence available to the reviewer in the event of queries. The coverage
achieved was 98% over the 60-min run. (b) An example of an automatically assigned peptide map of the molecule trastuzumab
by capillary electrophoresis electrospray ionization (CESI) separation with a color-coded assignment of the peptides identified