The Age of Big Proteins
Jeff Plomley

The Age of Big Proteins

Can liquid-chromatography-tandem mass spectrometry help us ride a new wave of biotherapeutics? Perhaps, but challenges remain.

In the days since scientists began growing “human” insulin in the laboratory, the world of protein therapeutics has increased both in scope and popularity. There are now drugs for most of the major diseases, and this nascent industry is poised to grow even more.

And why not? Given the diversity of function which proteins exhibit, from enzymes to hormones, this class of biomolecules represents a rational starting point for the development of novel therapeutic compounds, exemplified by monoclonal antibodies, interferons, interleukin-based products, and clot-busting drugs.

Although the $48 billion dollar protein-based therapeutics market is still a relatively modest one, comprised of only 130 licensed drugs, analysts predict sales could reach $141 billion by 2017 with the anticipation of new biotherapeutics.  

In response to this growth, demand is expected to increase for accurate and selective protein quantitation. Typically, that job falls to immunoassays, which have been the mainstay of large molecule quantitation. However, in instances where antibodies lack adequate specificity, immunoassays don’t always do a good job of detection. Other limitations of immunoassays can include:

  • an inability to distinguish between a parent drug and its metabolites, which differ by only a small chemical modification outside of the region recognized by the antibody
  • limited linear dynamic range for quantitation
  • time and cost required for antibody production
  • the use of large sample volumes

The Promise of LC-MS/MS
When an immunoassay is not well suited to the challenges of a particular protein, one alternative technique quickly gaining momentum is liquid chromatography (LC) coupled with tandem mass spectrometry (MS/MS), which relies, in part, on a discovery as old as the X-ray. For example, the measurement of serum thyroglobulin (Tg) is used to monitor patients after treatment for differentiated thyroid carcinoma (TC). Difficulty in using Tg as a biomarker for the recurrence of TC in many patients stems from the presence of endogenous anti-Tg autoantibodies (Tg-AAbs), which can interfere with immunoassays and cause false-negative results. Last summer, scientists demonstrated that the use of LC-MS/MS conferred a level of selectivity and specificity for Tg which overcame this limitation, meeting acceptance criteria for use in this clinical diagnostic application (Clin. Chem. 59 982 2013).

While LC-MS/MS is now a universal approach for a host of small molecule applications such as drug toxicology testing, newborn screening and endocrine testing, the improved precision and accuracy of the technique coupled with higher throughput and large dynamic range, is also seen as a potentially powerful tool in the development of new biotherapeutics.

With LC-MS/MS, assay selectivity and specificity are improved due to the orthogonal separation of proteins in the condensed state chromatographically, and in the gas phase by MS/MS. In the latter process, molecular ions of interest are isolated and fired into a collision cell containing an inert gas, most commonly nitrogen. Translational energy is converted to internal energy upon collision with the inert gas and this pinball effect results in the fragmentation of the molecular ion into progeny ions whose mass-to-charge ratio (m/z) and relative abundance represents a specific signature for the analyte of interest.
The Hurdles with LC-MS/MS
Still, given the complexity of protein structure, several challenges must be overcome in order for LC-MS/MS to become routine in biotherapeutic analysis. These challenges include:

  1. Identifying the right peptide. Most LC-MS/MS assays for proteins > 5 kDa are actually based upon quantitation of representative proteolytic peptides following enzymatic digestion of the target, rather than the target itself. Thus one of the major challenges involves the identification of a signature peptide whose sequence contains minimal homology with non-targeted proteins and can be formed reproducibly by the enzymatic digestion procedure, is sufficiently retained by LC, and easily ionized and dissociated during MS/MS analysis. Easing the process somewhat is the ability to predict candidate signature peptides in-silico. While these peptides can be compared against databases such as PeptideAtlas and Skyline to ensure a lack of interference from endogenous proteins, it is often difficult to predict ionization efficiency and MS/MS conversion efficiency in the absence of experimental data.
  2. Refining sample extraction. In general, the most sensitive LC-MS/MS assays involve depletion of highly abundant endogenous proteins or enrichment procedures using immunoaffinity techniques, whereas the least sensitive assays do not apply any purification prior to digestion.
  3. Separating the target peptide quickly enough. Time is of the essence. Chromatographically, fast and efficient separation of the target peptide is necessary to separate potential interferences while allowing high throughput. These requirements can be met using ultra-high performance liquid chromatography (UHPLC) techniques, including micro-flow and nano-flow chromatography.
  4. Finding the best MS technique. Currently, triple-stage quadrupole (QqQ) mass spectrometers are considered the gold standard for quantitation due to their high selectivity and sensitivity. Hybrid instruments incorporating ion trap functionality (e.g. AB Sciex Qtrap 6500) can offer further selectivity and specificity by leveraging an additional stage of fragmentation in the ion trap, giving MS/MS/MS.

While these challenges might seem daunting, instrument manufacturers continue to provide new technologies to facilitate large molecule quantitation. For example, further MS selectivity gains may be realized in alternative platforms such as the AB Sciex 5600 quadrupole time-of-flight (QqTOF) mass spectrometer, which allows quantitation based upon the exact m/z of progeny ions acquired at high resolution. New techniques using high field asymmetric waveform ion mobility spectrometry (FAIMS) allows ions of the same mass to be separated based upon molecular cross section, a useful strategy when facing difficult interferences. These and other evolving technologies are allowing the field to keep pace with the increasing demand for improved selectivity, sensitivity, and sample throughput.

And perhaps bring a new wave of biotherapeutics to market.


  1. Westinghouse Electric’s portable mass spectrometer, one of the first commercially available instruments, here being used for hydrocarbon analysis of petroleum products. Photo was taken in 1948. (ASMS)
  2. An AB Sciex QTrap 6500, one of the most sensitive triple-stage quadruple mass spectrometers on the market today. (AB Sciex)