What is mass spectrometry?

All mass spectrometers have three primary components; an ion source, a mass analyzer, and an ion detector. Samples are loaded into the mass spectrometer in either liquid, gas, or dried form and are then vaporized and ionized by the ion source. There are variations of these components in each type of mass spectrometer, which offer a diverse range of options for testing different physical properties of samples and data collection.

We will cover the basics on mass spectrometry; focusing on the history, technique, and the many applications of use.

Introduction to protein mass spectrometry

Originally developed 100 years ago, mass spectrometry was originally used to measure elemental atomic weights and the natural abundance of specific isotopes. Mass spectrometry was first used in biological sciences, in order to trace heave isotopes through biological systems.

In 1998, German physicist Wilhelm Wien laid the foundations of mass spectroscopy when discovering that charged particles could be deflected by a magnetic field. This was later applied by J.J. Thomson in the creation of the parabola spectrograph (the earliest version of a mass spectrometer).
In later years, mass spectrometry was used to sequence oligonucleotides and peptides, as well as in the analysis of nucleotide structures.
The mass spectrometry equipment of today is integrated with computer systems in order to generate data. A mass spectrum is the graph obtained by performing mass spectrometry, which details the relation between the mass to charge ratio and ion signal.

The development of macromolecule ionization methods, such as electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI), were monumental in the study of protein structure by mass spectrometry.

How mass spectrometry works

The underworking of mass spectrometry lies in Newton’s second law of motion. Using this property of matter, mass spectrometry plots ions of varying masses on a mass spectrum.

From the law, we can infer how much mass is relevant to the inertia and acceleration of a compound. Ions with different mass to charge ratios are thus deflected by different angles in an electric or magnetic field.

The history of proteomic workflows

Mass spectrometry has been incredibly useful in proteomics; the study of all proteins in a biological system (e.g. cells, tissue, organisms) during specific biological events. This is because mass spectrometry offers data on the quantities, functions, folding, and interactions of proteins.

Proteomics is a challenging field of study, as the dynamic nature of protein expression is so complex. Furthermore, the majority of proteins undergo some form of the posttranslational modification (PTM) leading to more challenges in genomic and proteomic studies.

Over the last 15 years, Mass spectrometry has been pivotal in the advancement of proteomics.

Mass spectrometry technique overview

When the molecules receive a charge, the mass spectrometer allows for the ions to accelerate throughout the mass spectrometer system. The ions encounter either electric or magnetic fields (or a combination of the two) via the mass analyzers. This then deflects the paths of the individual ions based on their specific m/z.

The mass analyzers include:

• Time-of-flight (TOF)
• Orbitraps
• Quadrupoles and ion traps

Each of which has their own respective characteristics. Mass analyzers can be used in either of two ways: to separate all analytes for analysis, or to be used like a filter that deflects only specific ions towards the detector.

Once ions have successfully been deflected via the mass analyzers, they come in contact with the ion detector. The detector emits a cascade of electrons when each ion hits the detector plate, resulting in amplification of each ion hit. This will improve sensitivity for detection.

These processes are performed under a high vacuum (10-6 to 10-8 torr), which removes contaminants from gas molecules, as well as neutral, and non-sample ions. By removing contaminants, you can mitigate the risk of these molecules colliding with the sample ions that may alter their paths and produce non-specific reaction products.

Mass spectrometers are connected to computer-based software that measure oscillation and frequencies of ions. This is done using image current detection. The program detects ions and organizes them by their m/z values and relative abundance.

Using established databases, these ions can then be matched and identified based on the m/z value.

Four main parts of mass spectrometry

Inlet system

One of the greatest advancements in mass spectrometry technology occurred with the implementation of the inlet system. In 1960 A. James and A. Martin first interfaced gas chromatography (GC) with a mass spectrometer. This was done by using a packed column injection.

This was later improved upon when Swedish medical scientist, Einar Stenhagen patented a high flow packed column with a jet separator and a large bore capillary column (530-750µm) that was connected to the mass spectrometer’s vacuum chamber. This advancement changed the sample flow rate into the ion chamber. This development would set the foundation for MS being such an effective tool for analytical chemistry.

Ionization

When processing a sample in mass spectrometry, the sample is bombarded by electrons. These electrons move between cathode and anode. As the sample passes through the electron stream, electrons with high energy knock electrons out of the sample and form ions.

Deflector

Deflection occurs as the ions interact with a magnetic field. The field deflects ions based on the ions' charge and mass. If an ion is heavy, or has two or more positive charges, then it is least deflected. Whereas, if an ion is light or only has a single positive charge, then it is deflected more.

Ion detector

The ion detector plays a key role in the analysis process. The ions with correct charge and mass will move to the detector, where the ratio of mass to charge is analyzed.

Protein mass spectrometry applications

There are numerous applications for mass spectrometry, including both qualitative and quantitative uses. Mass spectrometry is most commonly used in analytical laboratories for the study of physical, chemical, or biological properties of compounds.

Additional applications of mass spectrometry:

• Identifying unknown compounds
• Determining the isotopic composition of elements in a molecule
• Determining the structure of a compound based on fragmentation
• Quantifying the amount of a compound in a sample
• Understanding the fundamentals of gas phase ion chemistry

This makes mass spectrometry an ideal tool in drug discovery, clinical testing, genomics, geology, and environmental studies on food and soil. One of the more popular uses of mass spectrometry is in the carbon dating process used by geologists and other relevant fields.

Quality control via mass spectrometer

Ion detection is essential to science, as it provides an accurate methodology to understand the composition of a given sample, which can be directly applied to public health and safety.

Many companies will establish testing protocols throughout product development and the manufacturing process to uphold quality standards. Having this insight throughout each necessary phase of production can help determine issues of contamination, errors in process chemistry, or other anomalies that may present a risk to consumers.

Some contaminants in biopharmaceutical products could trigger immunogenic responses in patients, making it critical to detect and eliminate any adulterants present in a product.

Liquid chromatography MS (LC-MS) can detect peptides with incredible sensitivity (at the atomic level), offering promise for accurate product formulation, monitoring stability, and identifying impurities.

This level of accuracy is often credited to western blotting techniques like ELISA, which requires an antibody treatment technique. However, when a compatible antibody is unavailable, mass spectrometry can deliver highly accurate results without the use of antibody reagents.

Conclusion:
Mass spectrometry is an optimal analytical technique for countless applications, spanning multiple fields of science. Avantor offers the expert knowledge on the tech behind mass spectrometry, to support all of your laboratory and manufacturing needs.