Sensitive techniques for complex samples

Sensitive techniques for complex samples

The field of metabolomics is a key discipline in the study of the metabolism of living organisms in a wide range of conditions, including health and disease. The two main analytical techniques used for metabolomics include nuclear magnetic resonance (NMR) and mass spectrometry (MS), both of which allow for the detection of many different metabolites. The major strengths of NMR [1] are precise quantitation and superior compound identification; however, a major weakness of the method is in its low sensitivity (metabolites must exceed 1µM).

MS can typically detect in the femtomolar to attomolar range. Coupled to either gas chromatography (GC), ion chromatography (IC) or liquid chromatography (LC), MS can routinely analyze hundreds of compounds in a single sample and run, making it a very powerful and high-throughput process. With the advancement of high resolution accurate mass (HRAM) MS systems, as well as enhanced metabolite databases/libraries, metabolite identification has improved significantly.

Innovations in MS have also enabled metabolomics to emerge as its own field of study, and to complement genomics and proteomics (multiomics) as core technologies in academic and industrial research labs.

This overview outlines the role of mass spectrometry in the field of metabolomics and reviews MS methodology and instrumentation.

Introduction to mass spectrometry for metabolomics

What is mass spectrometry?

Mass spectrometry (MS) measures the mass-to-charge ratio (m/z) of ions to identify and quantify molecules in simple and complex mixtures. The development of high resolution accurate mass (HRAM) MS workflows with high throughput and quantitative capabilities has expanded the scope of what we know about the metabolites involved in different cellular and biological pathways; it has also enabled putative biomarker discovery in several related research areas.

How does a mass spectrometer function?

A mass spectrometer contains an ion source, a mass analyzer and an ion detector. The nature of these components varies based on the type of mass spectrometer, the type of data required, and the physical properties of the sample. Samples are introduced into the mass spectrometer in liquid or gas form and then vaporized and ionized by the ion source (e.g., ESI, APCI).

What is Mass Spectrometry?

Once ionized, the ions can be accelerated through the remainder of the system. Electric and/or magnetic fields from mass analyzers deflect the paths of individual ions based on their mass and charge ratio (m/z). Commonly used mass analyzers include time-of-flight [TOF], orbitraps, quadrupoles and ion traps, and each type has specific characteristics. Mass analyzers can be used to separate all analytes in a sample during global (or full scan) analysis, or they can be used like a filter (e.g., quadrupole, ion trap) to deflect only specific ions towards the detector.

Ions then hit the detector. Most often, these detectors are electron multipliers or microchannel plates, and they emit a cascade of electrons when hit. This cascade is amplified or multiplied for improved sensitivity. The entire process occurs under extreme vacuum (10-6 to 10-8 torr) so that contaminating gases, neutral atoms and molecules, and non-sample ions are removed. Such contaminants can collide with sample ions and alter their paths, they can also result in non-specific reaction products.

Newer orbitrap analyzer technology [2] captures ions around a central spindle electrode and analyzes their m/z values as they move across the spindle with different harmonic oscillation frequencies. Orbitrap technology can achieve extremely high sensitivity and high resolution accurate mass (HRAM) of obtained mass spectra. Orbitrap HRAM has several advantages for metabolomics studies.

Mass spectrometers are connected to computers with integrated software that analyzes the ion detector data and produces spectra that organize the detected ions by their individual m/z values and relative abundance. These ions can then be compared with available databases and libraries to predict their molecular identities based on their m/z values.

Tandem mass spectrometry and MSn

In metabolomics, single MS or full scan data is used for relative quantitation (profiling) as well as for searching through MS databases such as METLIN for metabolite identities.

However, when additional data are required for specific ions, tandem mass spectrometry (MS/MS) and MSn are used. During these approaches, a sample is injected into the mass spectrometer, after which it is ionized, accelerated through and analyzed by mass spectrometry (MS1). Ions derived from the MS1 spectra are then selectively fragmented and analyzed through a second stage of mass spectrometry (MS2) to generate the spectra of the ion fragments. 

This fragmentation occurs by a number of dissociation techniques. One method involves hitting the ions with a stream of inert gas, which is known as collision-induced dissociation (CID) or higher energy collision dissociation (HCD). Other methods of ion fragmentation include electron-transfer dissociation (ETD) and electron-capture dissociation (ECD). 

These fragments are then separated based on their individual m/z ratios in a second round of MS; as a consequence, MS/MS or MS2 (i.e., tandem mass spectrometry) has been performed. Some instruments utilize a single mass analyzer for both rounds of MS while others combine multiple analyzers.

Tandem Mass Spectrometry and MSn, Figure 1
Tandem Mass Spectrometry and MSn, Figure 2

In metabolomics, MS/MS and MSn are commonly used to increase confidence during metabolite identification when searching through MS/MS mass spectral libraries. With more advanced MS systems, multiple sequential rounds of mass spectrometry (e.g., MS3) can be achieved, and these data can be analyzed with ion fragment tree data to achieve further structural elucidation.

Selective reaction monitoring (SRM)  

SRM is the mass spectrometry approach of choice for routine quantitation of well characterized compounds. In this approach, MS/MS is typically used, and SRM experiments are employed using triple quadrupole mass spectrometers. Methods are developed to monitor compounds with a certain m/z and unique diagnostic ions specific to that compound for added confidence.

Selective Reaction Monitoring (SRM)

This approach allows compounds to be detected and quantified in complex matrices such as urine, plasma, blood and food, such samples are commonly analyzed in metabolomics experiments.

Parallel reaction monitoring (PRM)

This new method for quantitation takes advantage of the HRAM MS/MS capabilities found in Orbitrap-based mass spectrometers. PRM provides high selectivity, sensitivity and throughput quantitation, along with confident targeted confirmation. This method is best suited to quantifying tens to hundreds of targeted metabolites in complex matrices.

In PRM, the third quadrupole of a triple quadrupole is substituted with an HRAM mass analyzer, such as an Orbitrap, to detect all target product ions in parallel using one, concerted high resolution mass analysis. One of the main advantages of this approach includes less method development as compared to SRM analysis.

Parallel Reaction Monitoring (PRM)

  •  [1] Nicholson J, Connelly J, Lindon J et al. (2002) Metabonomics: a platform for studying drug toxicity and gene function. Nat Rev Drug Discov 1(2):153–161. PubMed
  •  [2] Hu Q, Noll RJ, Li H et al. (2005) The Orbitrap: a new mass spectrometer. J Mass Spectrom 40(4): 430–443. PubMed