The advent of novel ionisation techniques in mass spectrometry (MS), namely electrospray ionisation (ESI) and matrix-assisted laser desorption/ionisation (MALDI), and continued efforts to miniaturise sample handling procedures confront MS operators with new challenges: interfering compounds. Some are well-known from traditional analytical techniques and some are novel. These compounds may be intentionally added or inadvertently leach into the sample stream originating from the preparation environment and may include contaminated surfaces. Knowledge of the identity of these interferences is an essential first step for method optimisation and implementation of stringent quality control measures.

Over the past 20 years the introduction of novel ionisation techniques in mass spectrometry (MS), such as laser desorption/ionisation (LDI), matrix-assisted laser desorption/ionisation (MALDI) and ambient pressure ionisation techniques such as electrospray ionisation (ESI) or atmospheric pressure chemical ionisation (APCI), have revolutionised and enabled the so-called “omics”-research areas, including proteomics, metabolomics and lipidomics. The continued success and deepening impact of modern MS in biological research areas will largely depend on how readily this technique can be adopted and its use be expanded as a reliable and robust bioanalytical tool into mainstream laboratory processes. Stringent quality control mechanisms in terms of experimental setups, data processing and reporting, and standard operating procedures (SOPs) for good laboratory practices (GLPs) will gain importance, especially if modern mass spectrometry is increasingly applied in clinical research and routine diagnostics. One important aspect of quality control encompasses the awareness and knowledge of interfering chemical compounds that could negatively impact results from modern MS experiments.

With an increasing emphasis on miniaturised sample handling, the need to know the identities of interfering compounds will intensify accordingly. Since interfering compounds often enter the sample stream from contaminated or treated surfaces, increasing surface/volume ratios with the use of miniaturised sample handling technologies such as capillary columns or microfluidic devices will exacerbate potential interfering problems [Figure 1]. The dramatic increase of occupied surface area for a given sample volume with decreasing internal diameter of a capillary is a major limitation towards further miniaturisation efforts by employment of smaller inner diameter capillaries (< 10 µm) or smaller microfluidic channels. Besides potential sample loss due to irreversible binding to the surface, or orifice plugging by fine dust particles that are typically in the size range of 1-10 µm, leaching and potentially accumulating contaminants from the increased occupied surface pose major challenges towards the successful application of miniaturised sample handling technologies. Consequently, knowledge of identity and possible origin of potential contaminants is a mandatory first step to effectively cope with this challenge. 

A recent collaborative effort among colleagues from MS core facilities across Canada, including my own laboratory, provides a comprehensive summary of known interfering compounds often observed in modern MS applications [1]. The review/tutorial is organised in several sections dealing with both proteinaceous and non-proteinaceous compounds as the potential interferents in mass spectrometric analysis. Interfering compounds originating from proteins can typically be separated into two categories. The first category includes proteins or protein mixtures that are intentionally added during sample preparation, including proteases for subsequent peptide mapping experiments, proteins used for specific binding experiments, and auxiliary proteins or complex protein mixtures to eliminate non-specific binding to surfaces, (e.g. bovine serum albumin or whole serum). The second category includes proteins that are unintentionally added to the sample, the major class being human keratins which are omnipresent in lab and household dusts and can pose a significant challenge in bioanalytical MS if proper cautionary procedures are not followed.

Non-proteinaceous contaminants include polymeric compounds and plasticisers that have also been known to interfere in more traditional MS techniques, such as in gas chromatography-MS. Adduct formation with various cations, solvents or other molecules is more specific for the ESI and MALDI ionisation processes. A unique phenomenon in MALDI analysis is the formation of matrix clusters; their formation patterns can be predicted and due to their different fractional masses, can easily be discerned from biomolecule signals [2] [Figure 2]. Interfering compounds that are often deliberately added during sample preparation include buffers, dyes, detergents and other auxiliary compounds; the knowledge of their identity is essential to evaluate their potential interference in MS analysis.

As a supplement (available online [1]) we provide an Excel spreadsheet containing a database with more than 700 interfering ion signals that have either been reported in the literature or have been observed in our own laboratories over the past decade. We are aware that this list is dynamic and by no means complete. New contaminants or otherwise interfering compounds will continue to emerge, given that novel materials for sample preparation technologies are constantly introduced. However, we anticipate this database will provide a solid foundation, for many laboratories, that can easily be expanded by their own list of known and yet to be identified lab-specific interferences.

Other important classes of potential interferences (which we did not cover in our review) are modifications of analyte molecules that are induced by the sample preparation process or ambient environment conditions. One prominent example was recently described by Cohen and covers complex oxidation of biomolecules induced by ozone present in ambient air [3].

Knowing the identities of potential contaminants is not only valuable or necessary for the interpretation of interferences in MS studies, but is often essential for biochemical experiments, especially if interfering compounds have bioactivity and threaten to negatively impact the progress of an experiment. Recently this was strikingly documented by Andrew Holt and colleagues at the University of Alberta, where they found two additives originating from disposable laboratory plasticware (namely oleamide and di (2-hydroxyethyl) methyldodecyl ammonium) to interfere with kinetics experiments of human monoamine oxidase-B [4].

References
1. Keller BO, Sui J, Young AB, Whittal RM. Anal Chim Acta 2008; 627: 71-81.
2. Keller BO, Li L. J Am Soc Mass Spectrom 2000; 11: 88-93.
3. Cohen SL. Anal Chem 2006; 78: 4352-4362.
4. McDonald GR, Hudson AL, Dunn SMJ, You H, Baker GB, Whittal RM, Martin JW, Jha A, Edmondson DE, Holt A. Science 2008; 322: 917.

The author
Dr Bernd O. Keller
Assistant Professor
Department of Pathology
and Laboratory Medicine
Child&Family Research Institute
University of British Columbia
Vancouver, Canada
berndkel@interchange.ubc.ca

Bio-imaging MS techniques reveal molecular details about complex systems

Understanding biology at the systems level is difficult, especially when studying complex specimens like tissue slices or communities of organisms in a biofilm.
Scientists must be able to identify, quantify and locate the molecules present in the samples. At Georgia Tech, researchers from the Colleges of Sciences and Engineering have joined forces to create the Center for Bio-Imaging Mass Spectrometry (BIMS), which aims to tackle these types of challenges.
In the April 15 issue of the journal Analytical Chemistry, a research team including Merrill, Cameron Sullards, director of Georgia Tech’s Bioanalytical Mass Spectrometry Facility, and Yanfeng Chen, a research scientist in the School of Chemistry and Biochemistry, showed that the homogeneity of the matrix in MALDI/MS could be improved. With this development, broader categories of
compounds, such as lipids, could be analysed. The researchers used an oscillating capillary nebuliser to spray small droplets of matrix aerosol onto the sample surface – a process similar to airbrushing. Using histological samples provided by Timothy
Cox, a professor of medicine at the University of Cambridge, the researchers could profile and localise many different lipid species in the samples. Specifically, they localised sphingolipids that accumulate in the brain when there is a genetic defect. This research was funded by the National Institutes of Health. Facundo Fernandez, an assistant professor in Georgia Tech’s School of Chemistry and Biochemistry, recently used mass spectrometry techniques to detect counterfeit anti-malarial
drugs.While MALDI samples must be analysed in a vacuum, recent advances allow samples to be studied under ambient conditions. Fernandez has been using desorption electrospray ionisation (DESI). With DESI, a high-speed, charged spray
containing alcohol and water is directed at a sample a few millimeters away. The solvent droplets pick up portions of the sample through interaction with the surface and then form highly charged ions that can be analysed. Fernandez and his research team recently used DESI to analyse nearly 400 drug samples provided by public health authorities to identify counterfeit anti-malarial drugs. Activities aimed at addressing the widespread problem of counterfeit anti-malarial drugs were reported recently in the journal PLoS Medicine. Georgia Tech’s efforts to develop faster analytical techniques were sponsored by the U.S. National Science Foundation, while the sample analysis was supported by a small grant from the
World Health Organisation. In ovarian cancer research, little is known about how biomarkers and low-mass signalling molecules increase or decrease in abundance with treatment. Fernandez has teamed with Thomas Orlando, chair of the School of Chemistry and Biochemistry, to use DESI and laser desorption single photon ionisation mass spectrometry (LD/SPI-MS) to investigate this issue. Because the
two techniques overlap in mass ranges, using both provides a more complete investigation of the biomarker profiles. Because it does not use a matrix, LD/SPI-MS can detect low-mass molecules – such as sugars, amino acids, small peptides and cytotoxic compounds – formed as result of cancer treatment.
It could achieve higher spatial resolution and sensitivity than typical commercial mass spectrometers that rely on the laser desorption of ions.

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