Proteomics studies utilizing mass spectrometry have uncovered a pentabyte worth of information and provided key insights into a diverse array of targets. Proteomics researchers continue to push mass spectrometry (MS) limits, examining gross as well as very subtle variations to proteins, such as post-translational modifications or even individual ions. Manufacturers have responded by making technological improvements in three main components of MS systems - the ion source, mass analyzer, and detector.
Enhancements in ionization methods. To enable any means of characterization by MS, efficient ionization is necessary to convert molecules to measurable units of gas-phase ions. However, no single ionization method is capable of ionizing all components of complex mixtures to give a representative view of the molecular composition. In addition, sample preparation can be detrimental to converting solution or solid-phase molecules to gas-phase ions.
An important attribute of a novel ionization process for use in MS is its simplicity and flexibility to be hyphenated to conventional liquid-based separation methods. Liquid chromatography (LC) separation with ion mobility spectrometry (IMS) provides additional separation for gas-phase ions. The near-instantaneous separation afforded by IMS and MS reduces complexity, provides information about the size and shape of ions, and is applicable for material analysis even without prior separation using LC. The technology developments have potential of extending MS to areas of high-performance characterization using advanced fragmentation technology as well as to areas where cost, robustness, and simplicity are important assets such as clinical settings and field-portablity.
Improvements in detectors. During mass spectrometry analysis, after traversing the mass analyzer, ions are transmitted to the detector and are converted into a digital signal. Ionized peptides emerge from the analyzer and hit the detector plate, whereupon a cascade of electrons is emitted (usually via electron multipliers or micro-channel assemblies) and the signal is digitized. For TOF-based instruments, this ion-conversion step can be improved by making it faster, which improves resolution. Instrument companies are improving detectors, by widening the dynamic range. Dual-channel detection systems which add additional orders of magnitude of dynamic range compared with previous-generation detection systems have aided the analysis of complex protein samples.
High-performance mass analyzers. The recent development of high performance mass analyzers has enormously advanced mass measurement in terms of mass resolution and mass measurement accuracy. Additionally, structural characterization is enhanced using newer ion fragmentation methods like electron-capture dissociation (ECD) and electron-transfer dissociation (ETD), and interfacing IMS with MS provides inroads to the determination of structure (shape). These capabilities are facilitated by the production of multiply charged ions. Original equipment manufacturers are offering equipment that provide higher throughput, faster scan speed, wider dynamic range, more sensitivity, and higher resolution - all in less space. The compact unit is a bench-top system that has integrated all of the same technology as its larger counterparts. Building smaller MS equipment is often determined by the size limitations of the internal components. Reducing the size of components, including the capillary tubes and caps, equates not only smaller package sizes but also interchangeability.
MS - An Old Entrant in a New Cloak
When it comes to the field of commercial diagnostics, immunoassays have always reigned to be the technology of choice for decades. Multitude of analyzers are available in the market today, which are designed to perform immunoassays with pre-designed kits for utmost specificity and precision. However, for survival in the diagnostics business, though cost and quality of service are predominant factors, to grow and differentiate, it needs to be supported by technologies, which can widen the spectrum of testing services.
One such technology which has gained wide acceptance in diagnostics is mass spectrometry (MS). Though MS as a technology is not new and also its applications in the field of pharmaceuticals and food testing one well documented, MS with liquid chromatography (LC), gas chromatography (GC), inductively coupled plasma (ICP) are the technologies of choice for major diagnostic players in the clinical domain to invest in. With the major driving force being versatility, sensitivity, and precision offered by the platform, the range of biomolecules can be analyzed or have the feasibility to analyze multiple analytes in a single run.
With the major advantages of the technology listed, on the downside, the cost of the entire platform is a very huge investment for any laboratory, and becomes a major block for small-scale players from entering this domain. To sustain and support a laboratory workload, the analyzer mechanics also have to be sturdy requiring minimal maintenance and breakdown recovery time.
Having understood the major perils which might be needed to be addressed by the technology partners during service for diagnostics, changes and upgradations in the available platforms are being done in a dynamic fashion. To ensure uniformity, pre-designed kits for testing of analytes by MS have also started appearing in the market. These are inclusive of controls and calibrators, along with all the reagents for sample pretreatment to ensure variations due to manual intervention is minimum. Also, technology providers have gone a step ahead and are open to providing their method development teams service to aid in appropriate and robust protocol development.
With such personalized care and efficient service being doled out, and we as a major player in diagnostics experiencing this, have no qualms in stating that, an inter personal relationship to this magnitude will aid in strengthening the bond between diagnostics and technology. For an industry which thrives on innovations, MS is definitely an addition to enhance the portfolio of services offered.
Dr Sandhya Iyer
Thyrocare Technologies Limited
MS Applications in Clinical Microbiology
Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) offers the possibility of accurate, rapid, inexpensive identification of bacteria, fungi, and mycobacteria isolated in clinical microbiology laboratories. In 1994, Cain et al reported that MALDI-TOF MS could be used to differentiate selected bacteria by analysis of protein profiles from disrupted cells. The method as initially introduced is technically simple and rapid. Bacterial colonies are removed from agar culture plates, mixed with an excess of UV-absorbing matrix, and dried on steel target plates. The dried preparations are exposed to laser pulses, resulting in energy transfer from the matrix to the non-volatile analyte molecules, with desorption (removal) of analyte into the gas phase. The ionized molecules are accelerated by electric potentials through a flight tube to the mass spectrometer, with separation of the biomarkers determined by their mass/charge ratio (m/z: z typically is 1). The profile of biomarkers is then compared with the profiles of a collection of well-characterized organisms in database.
Mass spectrometry via MALDI-TOF has revolutionized organism identification in few minutes at less than Rs.20 per isolate. It is a path breaking technology capable of identifying bacteria, mycobacteria, yeast , molds, anaerobes, and NTM with a prerequisite of a pure isolate on a solid agar. Limitation is it cannot be applied directly to those specimen which contain more than 1 isolate nor to identify viral and parasitic organism as of now. Research is happening in the field of virology where this will be possible down the line.
Further application on antimicrobial sensitivity has already happened where it facilitates random detection of ESBL and carbapenemase enzyme.
Most biomarkers detected in MALDI spectra are intracellular proteins primarily in the range of 4000 to 15,000 Da. These are highly conserved housekeeping proteins and serve as ideal biomarkers for characterizing individual species. In general, more than 90 percent of all isolates are identified at the species level, 98 percent are identified at the genus level, and less than 1 percent are incorrectly identified. The most common reason an isolate is not identified is because it is not included in the database. Misidentifications are most commonly observed with taxonomically related bacteria, such as Shigella with Escherichia and Streptococcus pneumoniae with S. mitis.
MALDI-TOF MS offers the possibility of accurate, rapid, and inexpensive identification of microorganisms. The procedures for preanalysis processing of organisms and analysis by MALDI-TOF MS are technically simple and reproducible, and commercial databases and interpretive algorithms are available for the identification of a wide spectrum of bacteria, yeast, molds, and mycobacteria.
Dr Shalabh Malik
HoD-Microbiology & Clinical Pathology
Dr Lal Path Labs Private Limited
Advances in instrumentation in the past 2-3 decades have rendered MS an important tool for structure elucidation, quantification, and revelation of biological consequences of DNA adducts. The applications of MS-based DNA adduct analysis are used for predicting the therapeutic outcome of anti-cancer agents, for monitoring the human exposure to endogenous and environmental genotoxic agents, and for DNA repair studies. In addition, competitors are looking to adopt the general life science trend of miniaturization to bring the technology to lower-budget laboratories. A combination of collaborative activities across different laboratories and multicenter studies will aid in establishing MS imaging as a proven and reliable technique in biomedical and bio-analytical applications in the future.