Groundbreaking research by physicist J.J. Thompson in the early 1900s led to development of the first mass spectrometer in 1918. Early instruments enabled the discovery of new isotopes of common elements such as magnesium, mercury and chlorine.

Governments dominated mass spectrometry (MS) for the next 30 years in the service of the war effort and the development of early nuclear weapons. The next phase of MS development, roughly between 1950 and 1990, was characterized by the entry of companies introducing MS instruments for the petroleum and chemicals industries, and later for pharmaceuticals. Still, MS remained a specialist discipline until microelectronics came into its own during the late 1980s.

Within the last 30 years MS has undergone revolutionary changes. In 1990, instruments cost many hundreds of thousands of dollars, took up the better part of a lab’s floor space, were operated by dedicated, doctoral-level staff and existed primarily within core facilities. Chemists would prepare pure samples, “send them down” and hope to receive a simple spectrum several days later.

Enabled by advances in materials science, microprocessors/data storage and software languages, the democratization of MS is fully underway and in fact entering a stage of relative maturity. Instrumentation is still expensive but only a fraction as costly, in real dollar terms, as it was during the late 1980s. Room-sized systems have shrunk to the point where we now use bench-sized “detectors” for front-end separations like gas chromatography (GC) and, importantly for biologists and biomedical researchers, gel electrophoresis and HPLC.

Companies might still employ Ph.D.-level mass spectroscopists, but demand for such experts has fallen because of the automation of MS methods and instrumentation, and overall greater user-friendliness. Spectroscopists’ skills are now applied to developing high-value methods or running complex experiments. In contrast to stand-alone systems for confirming molecular weights and simple, characteristic fragmentation patterns, modern mass spectrometers are more frequently sold as components of complete analytical systems used to address complex scientific problems.

Mass spectrometers work by determining masses of charged atoms and molecules. All systems consist of five principal components: an inlet, source region, mass analyzer, detector and data system [1]. Samples are introduced through the inlet either manually or through a suitable separations or injection system. Species ionize within the source, from which they are analyzed and detected under high vacuum. Today’s mass spectrometers use computer interfaces and specialized software to capture and interpret data. In addition, instruments used in the life sciences are often sold with the appropriate analytical front end, which separates molecules of interest from each other.

Sample preparation is critical for removing high-abundance species from highly complex biological fluids and extracts, for example albumin from blood. Sample prep has nearly become a separate “component” of mass spectrometry with emerging interest in the ‘omics sciences, which demand high sensitivity toward very low-concentration target species.

For life scientists, MS almost always means LC-MS or GC-MS. Stand-alone mass spectrometers, lacking an analytical front end, are still used for the analysis of pure solid and liquid compounds or as chemical-specific analyzers that don’t require separation from each other or the matrix in which they occur.

This MS buyer’s guide

Because of the enormous diversity in instrumentation, capabilities and applications, no buyer’s guide of modest length can serve as an exhaustive treatise on MS or answer every question related to “how to purchase a mass spectrometer.” Our goal is to provide an overview of the major components of an MS system and share some insights on the applications in which MS can accelerate scientists’ research projects.

In this guide, you will learn about:

  • MS instrument types and the components that make up the systems.
  • Molecules amenable to MS analysis.
  • Emerging applications.
  • A researcher’s perspective on the future of MS.

Depending on your level of expertise, you may find some sources of technical information more useful than others. Vendors are arguably the No. 1 information source for purchasers of instruments as diverse and complex as mass spectrometers. All MS manufacturers today maintain a treasure trove of published articles, white papers, unpublished results, side-by-side instrument comparisons, application notes and case studies. Top vendors welcome you to “kick the tires,” and many will accept difficult samples to demonstrate their instruments’ capabilities. Product managers, who often hold advanced degrees in spectroscopy, are eager to demonstrate their expertise and their companies’ capabilities. We also encourage you to visit Biocompare’s product directory to view some of the latest offerings in MS systems, reagents, accessories and sample-preparation tools.

MS system components

The guts of modern MS instruments are: an ionization chamber, mass analyzer and mass detector.

Most descriptions of mass spectrometers are named by combining the ionization method and the analyzer, for example MALDI (an ionization method) TOF (the analyzer).

Note that even within one instrument type, manufacturers have created additional component categories and subcategories. Vendors have also created application-specific MS-based workstations in which the components are optimized for a particular type of analysis, for example genetic analysis, small molecules, proteins, lipids, etc. Systems are increasingly specified and provided according to narrow analytic capabilities, and the required software often comes pre-packaged. Here is a brief listing and description of the major MS components.

Types of ionization (ionization chamber)

Electron Impact Ionization (EI)

  • Loss of electron leads to formation of positive ions
  • Considered "hard" ionization, in which high quantities of residual energy from the subject molecules results in high degrees of fragmentation
  • Useful for compounds with reasonable vapor pressures, which can exist as gases within the ionization chamber
  • Often generates a molecular ion peak as well as predictable fragmentation patterns used to confirm compound identities
  • Compounds < 1000 Da

Electrospray Ionization (ESI)

  • This “soft” ionization method provides multiply charged ions, particularly for peptides, proteins and nucleic acids
  • Little or no fragmentation
  • Compatible with compounds that are sufficiently basic or acidic in the gas phase to accept or lose protons; ionic compounds
  • Produces simple spectra without extensive fragmentation, which limits library-based identification
  • Compounds < 1000 Da

Atmospheric Pressure Chemical Ionization (APCI)

  • Works on compounds not sufficiently polar for electrospray
  • Uses a corona discharge to add a proton, forming positively charged species
  • Variant desorption chemical ionization (DCI) reduces chemical degradation, but results are not reproducible
  • Another variant, negative ion chemical ionization (NICI), is used for compounds capable of forming negative ions
  • Compounds less than approximately 1,000 Da (DCI), 1,500 Da (NICI) and 2,000 Da (APCI)

Laser Desorption

  • Soft ionization technique for delicate samples
  • Matrix-assisted laser desorption ionization (MALDI) is the most prominent
  • SELDI (surface-enhanced laser desorption ionization), a variant of MALDI, is frequently used in top-down proteomics
  • Ionization occurs through vaporization and ionization by a high-energy laser pulse
  • Applicable to large molecules like proteins, peptides and polymers
  • Samples may be introduced as is, with little or no sample preparation
  • Compounds of up to about 500,000 Da

Inductively Coupled Plasma (ICP)

  • Uses plasma ionization
  • For detecting metals, for example in metalloproteins
  • Compared with atomic absorption spectroscopy, ICP-MS is faster, less expensive and more sensitive
  • Used mostly for inorganic samples, but has been applied to detect metals in pharmaceutical products and, with size exclusion methods, for analysis of metalloproteins [2]

These ionization methods are common categories that manufacturers might mention when describing their systems. Literally dozens of variations exist on these general ionization types. Each has unique advantages and disadvantages in terms of sensitivity, molecular weight, usability and types of compound analyzed. We encourage you to review Biocompare’s mass spectrometry product-directory listings for system options available to researchers. Determining which method best suits your application involves consulting the literature, asking plenty of questions and requesting sample analyses.

Mass analyzers

The mass analyzer is the heart of an MS system. Analyzers are the component that separates ions according to mass-to-charge ratio. Mass analyzers come in several general categories but, like mass detectors, numerous variations are available for each. Similarly, some mass analyzers are better suited to certain applications than others.

Mass detectors receive species from the analyzer and report results based on an ion’s charge or momentum. Detectors typically amplify the signals, and some are thus capable of picking out single molecules.

Scanning mass analyzers are the mass equivalent of spectrophotometers, which separate light based on wavelength. In a scanning mass analyzer, a magnetic field is applied to a mixture of ions, which separate based on their mass-to-charge ratio. One variant, magnetic sector MS, fits the spectrophotometer analogy best. But because particles have different energies, these instruments use an electric sector to pre-select ions according to their energy. Scanning mass analyzers produce classical MS spectra with the highest resolution, quantitation, sensitivity and dynamic-range capability. They provide accurate mass measurements, but they are larger and more expensive than alternatives.

Quadrupole MS uses charged rods to filter ions of specific charge/mass ratios. Like magnetic sector instruments, quadrupoles (or “quads”) provide classical spectra with good reproducibility at relatively low cost. But their resolution is limited and peak height to mass must be tuned to obtain accurate quantitation. Quads are not suited for “pulsed” ionization methods like MALDI.

Time-of-flight (TOF) analyzers, as their name implies, measure the time it takes an ion to travel from the ion source to the detector. Because the starting time is critical for this calculation, ions should form via a pulsed technique like MALDI or rapid switching of electric fields. TOF instruments are the fastest mass analyzers with the highest mass range capabilities, which makes them suitable for gas chromatography as well as HPLC front ends. Shortcomings include limited dynamic range and precursor selectivity for MS/MS methods. TOF instruments are commonly used for biological samples, especially with MALDI and ESI [3].

Ion trap analyzers use a combination of electric or magnetic fields to keep ions within a defined space. Trapping of charge particles can be carried out by various methods— Penning trap, Paul ion trap and Kingdon trap, that differ in their procedures, but all are used to contain ions which will be detected and converted to a mass spectrum. Quadrupole ion traps use mass-selective ejection, in which trapped ions leave the analyzer in order of increasing mass. Ion traps are suitable when high sensitivity, rapid scanning and full spectra are required, for example with unknown samples and rapid HPLC separations [4]. The orbitrap is an improved modification of the Kingdon ion trap and was commercialized by Thermo Fisher Scientific over 10 years ago.

Tandem mass spectrometry (MS/MS or MS2) involves two stages of mass analysis, with a fragmentation step in between. The fragments reveal properties of the precursor ion unavailable through one-dimensional analysis [5]. Several types of tandem MS are available, including triple-quadrupole, quadrupole-TOF and ion trap-Fourier transform MS. Analytes include oligosaccharides, glycolipids and peptides/proteins.

Molecule types

MS handles a wide variety of analytes, ranging from ceramics to cells—and everything in between. Applications within the life sciences are similarly varied, covering every conceivable molecule type.

Proteins, polypeptides and individual amino acids are of great interest in proteomics. MS resolves many of these species through their fragments or as intact molecules. Note that in natural systems, proteins and some longer peptides may undergo post-translational changes that include glycosylation, deamidation, phosphorylation and other modifications. Often prior to loading on an MS for analysis, these molecules are subjected to 2D gel electrophoresis or HPLC. MS systems for proteomics are based on ESI and MALDI ionization and use triple-quadrupole, ion trap or quadrupole-TOF analyzers.

Oligonucleotides (genes or gene fragments, both naturally occurring and synthetic) are complex molecules consisting of a nitrogen-bearing ring structure, ribose sugar and phosphate. Their chemical complexity makes MS a preferred analysis method for identification and sequencing. Oligos are analyzed through electrospray ionization or MALDI-TOF mass spec [6].

Oligosaccharides (or sugars, glycans, polysaccharides) are ubiquitous in nature and are often conjugated to peptides and proteins. Glycan type and distribution are considered key quality attributes of therapeutic proteins. Determining the sequence of polysaccharides is complicated by the large number of chemical and stereochemical isomers of the building-block sugars and their linkages. Extensive branching also complicates analysis. HPLC separation followed by MALDI-TOF is the method of choice for glyans, although these methods are not as mature as for other biological molecules [7].

Lipids comprise an extensive class of mostly carbon-bearing molecules with broad physiologic relevance. Lipid biosynthesis and metabolism are key indicators of biological processes. Lipidomics, the study of an organism’s lipid profile, is typically conducted using electrospray ionization sources and triple-quadrupole spectrometers. MS determines a lipid’s molecular weight, chemical composition and branching. A great number of MS instrument and system choices are available for lipids, including GC- and LC-MS, ESI-MS, ESI and MALDI.

Metabolites are molecules of low molecular weight (typically < 1800 Da) showing a wide variety of chemical and atomic compositions. Biologists and biomedical researchers are particularly interested in metabolites, because their relative concentrations provide a real-time snapshot of the state of the organism. Because of the high-molecular-weight dynamic range (1015) in metabolomics studies, sample preparation and high sensitivity are essential. Ionization methods for metabolomics include electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), atmospheric pressure photoionization (APPI) and fast atom bombardment (FAB) [8].

Emerging MS applications

The molecule types listed earlier attest to the applicability of MS analysis to a wide variety of organic molecules. End users need no further explication to recognize these compound types within their workflows.

With the evolution of MS and the introduction of novel capabilities, exciting applications have emerged that were impossible or inconceivable just a few years ago. Most involve the life sciences.

Metabolomics

Metabolomics was originally conceived as an academic research tool. Today, with LC-MS and appropriate methods routinely available, its application to drug discovery and development has been nothing short of remarkable.

By identifying the metabolomic profiles of physiologic states—diseased vs. healthy, treated vs. untreated, low-dose vs. high-dose—clinical researchers can determine if a drug is acting as intended. 

Comparisons between drugs may now be quantified through metabolomics instead of by statistical analysis of what are often subjective symptoms. The use of metabolomics to identify new drug targets and mechanisms of action, customize treatments and monitor treatment outcomes is now routine [9]. After a medicine is approved, metabolite patterns can measure a drug’s efficacy and toxicity through metabolomic biomarkers, sparing patients from unnecessary or ineffective treatments.

The metabolome is also a key entry point to systems biology, which seeks understanding of the interplay between components of complex biological systems [10]. At this level of sophistication, however, lack of software tools represents a significant bottleneck to systems-wide utility of metabolomic data. In addition to data processing and analysis, another significant bottleneck is method development/optimization.

Pathogen diagnosis

Hospital-acquired infections are a leading cause of illness and mortality in the United States, with 722,000 cases and 75,000 deaths shared in a 2011 survey [11]. Patients can become gravely ill before results from cultured specimens, considered the gold standard at the time for diagnosis, become available. Moreover, bacterial sequencing can be expensive and time- and effort-consuming. Patients are therefore automatically treated, as a preventive measure, and often with an inappropriate antibiotic. MALDI-TOF MS can identify common hospital-acquired pathogens in minutes instead of days. Thanks to its soft ionization mechanism, the technique identifies bacteria and their subtypes from blood, urine, stools and solid tissue samples [12].

Of great importance to public health is the application of MALDI-TOF to food and water samples, putative biological weapons and in environmental bacteriology. Because of its specificity, the technique easily distinguishes between Staphylococcus aureus strains that are susceptible and resistant to common antibiotics. In some instances, MALDI-TOF could replace culturing and PCR for diagnosis of bacterial and viral infections.

A barrier to adopting MS in clinical settings is demonstrating to the U.S. Food and Drug Administration (FDA) that tests have been validated in real-world clinical settings. All major MS instrument vendors have taken up the challenge, including Agilent, BioMérieux, Bruker, Sciex, Shimadzu, Thermo Fisher Scientific and Waters.

Drug and ingredient authentication

Using MS to test for components of cell-culture media (nutritional components, pesticides, adulterants and ingredients) is well established. Methods for detecting adulterants such as sugar substitutions, oils used in surfactants and detergents, excipients and pharmaceutical ingredients are similarly well established. Because of the chemical nature of these species, GC serves as the front end more frequently than in most other life sciences analyses.

The development of isotope-ratio tests for authentication of country or region of origin, ingredient sources and even process conditions began with archeology (carbon dating), took hold in the food industry and is now being adopted by life sciences suppliers and, with FDA approval, drug companies [13].

Isotope-ratio analysis relies on the peculiar fact that ratios of stable isotopes, for example 13C/12C, differ at various locations around the globe. The phenomenon arises from small but measurable isotope effects for compounds containing hydrogen, carbon, oxygen, nitrogen and sulfur, which are either contained in clouds or carried via weather conditions. Mass spectrometers that resolve molecular weight differences at concentrations common for nonmodal isotopes can therefore pinpoint a product’s region of origin. They are increasingly used for detecting counterfeit drugs, authenticating ingredients’ regions of origin, obtaining patent protection and isotopic fingerprinting [14].

Many types of MS analyzers capable of high-mass resolution are appropriate for isotope-ratio analysis. Some vendors have assembled systems dedicated to this application, and many more offer methods. Systems may use a combustion reactor, often combined with GC, as the analytical/sample-prep front end.

In the forefront

MS imaging has become popular through MALDI ionization and application of other softly ionizing methods [15]. The technique generates 3D volumes of molecules of interest, for example metabolites and proteins. Alternative ionization methods include ambient ionization techniques such as desorption electrospray ionization and matrix-assisted laser desorption electrospray ionization (a variation on the MALDI theme).

Top-down proteomics identifies, characterizes and sequences proteins in their intact form; conventional MS-based sequencing uses protein digests [16]. The advantage of the top-down approach is it provides a holistic view of protein translation, with all its inherent variability of post-translational modifications. This method requires a robust HPLC separation mode that retains the protein’s characteristics throughout the run. A recent advance in this area has been the introduction of less-hydrophilic hydrophobic interaction HPLC columns [17].

SPR-MS combines surface plasmon resonance (SPR) with MS to provide characterization of biomolecular interactions through SPR, followed by verification of the specific molecules that bind. The technique uniquely marries SPR detection of low-level molecular events with rigorous chemical confirmation [18].

A view from an expert

Philip Lorenzi, Ph.D., is co-director at the M.D. Anderson Cancer Center’s Proteomics and Metabolomics Core Facility in Houston, Texas. He and his colleagues have published extensively on MS within the life sciences, and he offers some insight into bottlenecks, challenges and what to look for when selecting or potentially purchasing MS instrumentation.

Lorenzi’s experience resides primarily with metabolomics, but his facility engages in both proteomics and metabolomics, two fields with similar challenges. He identifies method development and optimization as a key bottleneck. “To my knowledge, there aren’t any shortcuts to method development and optimization. You still need staff who are properly trained in method development and optimization, and it helps if they have strong chromatography experience.” 

The sheer volume of information generated by today’s life sciences experiments leads to hurdles in processing and analyzing data, as well. 

“Metabolite annotation—the identification of unknowns—remains a huge bottleneck in nontargeted metabolomics.”

Inconsistency of results has always plagued biology and biomedical research. This carries over to MS experiments, as well; responses can vary dramatically even among MS instruments of the same model. “You can run the same sample on two identical instruments, and you will probably see different responses,” Lorenzi says. “A friend recently told me that his group could not reproduce results using identical instruments in two different laboratories. Veterans of MS are intimately familiar with that phenomenon.”

A related issue involves bioinformatics software. Available software has problems that many users aren’t aware of, Lorenzi says. “Bioinformaticians and software engineers on my team identified a variety of errors in metabolomic data processing with existing software and are working with vendors to resolve the problems. This is a significant challenge, because most laboratories don’t have bioinformatics and software engineering expertise on staff and are therefore unaware of the problems.”

As for purchase decisions, Lorenzi recommends building in redundancy with other MS systems that already exist in your lab, so that if one instrument goes down for an extended period, methods may be ported to the backup system. This assumes, of course, that inconsistencies have been ironed out.

“All vendors and instrument models have strengths and weaknesses, but what is perceived to be a strength by one buyer may not be a strength for another buyer,” Lorenzi warns. “For example, a contract research organization has significantly different needs than an academic core facility, even if the work is ostensibly very similar. At a minimum, a responsible purchaser should conduct a due-diligence campaign that compares the performance of candidate instruments with a specific application.”

Finally, there are issues with throughput and availability, which for an expensive asset like a mass spectrometer can be substantial. “Especially for LC-MS systems, if the laboratory has a significant bottleneck associated with instrument time, one should consider a multiplexing solution that can potentially save significant time with targeted assays by implementing a switching valve and a scheduling queue to run samples in parallel,” Lorenzi says.

Closing thoughts

Mass spectrometry continues to evolve as a powerful methodology and analytical tool for detailed analysis of particles and molecules. The applications for this technology span all areas of life sciences, including basic discovery research projects, food and environmental testing, clinical applications, material sciences and drug and biotherapeutic development. 

At the sixty-fourth annual American Society for Mass Spectrometry (ASMS) meeting in San Antonio in which Biocompare was in attendance, a widely resonating theme from all instrument, reagent and software providers was bringing MS to all researchers—de-mystifying the “black-box” perceptions of MS and engaging scientists to collaborate, share and adopt the technology and create a more all-encompassing workflow that addresses their research challenges.

Tool providers will continue to push the limits of sensitivity and detection, but they are clearly focused on balancing these changes with ensuring a positive user experience with the instrument, reagents and software. This will be an ongoing process that will continue to uncover exciting new research findings.

Summarizing MS in its totality is beyond the scope of this article. Our intent is to provide an overview of MS system components, targets and emerging applications, as well as the views and perspectives of an MS researcher. We strongly suggest that you view Biocompare’s product directory for some of the latest MS offerings and that you consult the various tool providers to obtain details on which MS system will best address your research needs.

References

[1] Van Bramer, S, ”An introduction to mass spectrometry.” 
[2] Lothian, A, et al., “Standards for quantitative metalloproteomic analysis using size-exclusion ICP-MS,” J Vis Exp, 2016 Apr 13 (110). Pubmed link. [PMID: 27167680] 
[3] Kore Technology, “Introduction to time-of-flight mass spectrometry.” 
[4] Agilent, “Frequently asked questions about the Varian 500-MS ion trap mass spectrometer."
[5] National High Magnetic Field Laboratory, “Tandem mass spectrometry (MS/MS).” 
[6] Integrated DNA Technologies, “Mass spectrometry analysis of oligonucleotide syntheses.” 
[7] Sigma-Aldrich, “Mass spectrometry of glycans.” 
[8] Zhou, B, et al., “LC-MS based metabolomics,” Mol Biosyst, 8(2):470-481, 2012. [PMID: 22041788]
[9] Wishart, D, “Emerging applications of metabolomics in drug discovery and precision medicine,” Nature Reviews Drug Discovery [PMID: 26965202] 
[10] Aretz, I, et al., “Advantages and pitfalls of mass spectroscopy based metabolome profiling in systems biology,” Int J Mol Sci, 17(5):632, 2016.[PMCID: PMC4881458]
[11] Centers for Disease Control and Prevention, “HAI data and statistics.” 
[12] Singhal, N, et al., “MALDI-TOF mass spectrometry: an emerging technology for microbial identification and diagnosis,” Front Microbiol, 6:791, 2015 [PMID: 26300860]
[13] Jasper, J, “Using stable isotopes to authenticate pharmaceutical materials,” Pharmaceutical Security. 
[14] Isotech Laboratories, “Isotopic fingerprinting of solids and liquids.”
[15] Seely, E, et al., “3D Imaging by Mass Spectrometry: A New Frontier,” Anal Chem, 84(5):2105-2110, 2012.[PMCID: PMC3296907]
[16] Perkel, J, “Top-down proteomics: turning protein mass spec upside down,” Science/AAAS Publishing Office, September 11, 2015. 
[17] Chen, B, et al., “Online hydrophobic interaction chromatography-mass spectrometry for top-down proteomics,” Anal Chem, 88(3):1885-1891, 2016. [PMID: 26729044]
[18] Zhang, Y, et al., “Interface for online coupling of surface plasmon resonance to direct analysis in real time mass spectrometry,” Anal Chem, 87(13):6505-6509, 2015.[PMID: 26067340]