Living cells are composed of diverse biomolecules. Whether abundant or rare, these molecules help maintain cell functions and keep our bodies healthy. Changes in the amount of a biomolecule can also affect how well our bodies operate. Hence, researchers advance biological research by identifying existing and novel molecules that contribute to health and disease. These molecules are called biomarkers.
But what has driven the surge in biomarker research? The answer lies in mass spectrometry (MS).
MS analysis characterizes small molecules as well as larger molecules such as proteins at a high-throughput rate. MS’s capabilities have spearheaded the wholesale characterization of proteins and metabolites in samples. Known as proteomics and metabolomics, researchers can now choose from one of many mass spectrometers to profile biomolecules. Even so, MS is still time-intensive and costly since owning and maintaining a mass spectrometer can be expensive. The steps involved with tissue culture processing are also just as complex.
In this article, we will discuss the basics of MS and walk through the MS pipeline as it pertains to tissue cultures, a type of biological sample. We will then discuss how MS helps researchers use tissue cultures to advance clinical research. Finally, we will show how our leasing program helps you leverage the power of MS as you develop your biomarker discovery workflows.
What Is Mass Spectrometry?
MS is a technique that measures analytes, any compound of interest. These include proteins, amino acids, lipids, fatty acids, complex carbohydrates, and simple sugars.. The mass spectrometer identifies each analyte once it separates them by their chemical characteristics. All mass spectrometers contain four components:
- Inlet system: The inlet system introduces the sample into the mass spectrometer. It can be as simple as a port where the sample is injected. More commonly, intel systems contain an apparatus that separates compounds by chemical characteristics, such as a liquid chromatographer for organic compounds. An inlet system can handle gas, liquids, or solids. Which state of matter they handle distinguishes between the different kinds of mass spectrometers that exist.
- Ion source: All mass spectrometers have an ion source that performs the ionization step. Here, the ion source adds charges to the analytes. Ionization facilitates the separation of the ions by molecular weight and size of charge in the subsequent steps. Many kinds of ion sources exist, including electrospray ionization (ESI), matrix-assisted laser desorption/ionization (MALDI), and chemical ionization (NCI).
- Mass analyzer: The mass analyzer separates the ionized molecules to analyze their molecular weight. Quadrupoles are the most common component of the mass spectrometer, providing a mass filter of four parallel metal rods. Along with other mass analyzers such as a time of flight (TOF) analyzer, these devices assess molecular weight by measuring the time needed for an ion to bend in response to an electric field. Lighter molecules travel farther along the analyzer than the heavier ones.
- Ion detection system: After ion differentiation, the ion detection system measures ion abundance and sends the results into a data system. Multiple kinds of detection systems exist, but the electron multiplier (EM) detector is the most common. EM detectors can amplify individual ions by beginning a cascade that excites more electrons to make the ions detectable.
Researchers can also select from three types of MS depending on the kinds of biomolecules being profiled:
The Mass Spectrometry Pipeline: From Cell Culture to Data Analysis
Mass spectrometry provides a useful tool for characterizing cultured cells’ chemical composition. However, undertaking such a study features a multi-step process that must be conducted carefully.
- Sample preparation: Any mass spectrometry assay with tissue cultures begins with preparing the cultured cells. It begins with an incubation period where the cells are cultured under specific growth conditions. Tissue cultures can grow in free-floating suspensions or as adherent cells. For the latter, scientists must use enzymes that detach the cells from the surfaces on which they attach to harvest them. After obtaining the cells, centrifugation separates the cells from the liquid supernatant, enriching the cell fractions for the next step.
- Cell lysis: After harvesting the cells, lysis must occur. Lysis causes the cells to release the metabolites and proteins for quantification. Various buffers and detergents can adequately lyse the cells. Irrespective of the reagents used, researchers must be careful not to introduce contaminants incompatible with the mass spectrometer. These contaminants can obfuscate any molecular signals coming from the sample.
- Sample extraction: Once the lysates have been prepared, the proteins or metabolites of interest are extracted from the samples. Regardless of the extraction method employed, researchers must carefully consider how their extraction method contributes to the variability of the resulting proteomes.
- Molecular separation: After the specific kinds of biomolecules have been extracted, the biomolecules are commonly separated before ionization. Chromatography represents the first step for separating proteins and other biomolecules. Solvents such as acetonitrile and ammonium-based solvents provide the vehicle for separating these molecules at the liquid and gas phase.
- Data collection: After separating the molecules from a cell culture lysate, mass analyzers separate the ionized biomolecules by their mass-to-charge (m/z) ratios. Doing so allows researchers to identify biomolecules and/or proteins by their molecular weight.
- Data analysis: Mass spectrometers have an ion detection system that produces a dataset of relative abundances for each molecule at specific m/z ratios. Analyzing this data will depend on what data acquisition method the researchers used. These are split into two types:
- Data-dependent acquisition (DDA): In DDA, a full scan is first conducted. Then, the mass spectrometer selects precursor ions from the full scan for further fragmentation. This increases the resolution (res.) by obtaining cleaner spectra of the ions present in the samples at a given molecular weight range.
- Data-independent acquisition (DIA): DIA substantially increases the spectra of precursor molecules within an MS scan. Here, all precursor ions within the spectra are fragmented and classified into isolation windows. This technique is now possible for MS thanks to the increased sensitivity of existing mass spectrometers.
Applications in Proteomics & Metabolomics Research for Cell Cultures
MS is an essential tool for obtaining high-throughput and high-resolution metabolite and protein profiles. Such assays provide valuable insights into protein-protein interactions at the cellular and subcellular levels.
Researchers can also harness the power of MS as they evaluate three-dimensional (3D) cell cultures for studying disease and developing pharmaceuticals. Known as organoids and spheroids, they are derived from stem cells and mimic a given organ's key functional and structural aspects.
Mass Spectrometer Leases for Your R&D
Excedr is, first and foremost, a scientific equipment leasing company. Suppose you want to advance your proteomics and metabolomics research but find the upfront costs of buying a mass spectrometer too expensive for your budget. In that case, we can lease you one instead, helping you preserve working capital and extend your cash runway with significantly lower upfront costs and manageable monthly payments.
We do not carry an inventory but instead acquire the exact mass spectrometer you want when you need it from the most well-known vendors—or any manufacturer you choose. Here are just some of the vendors we work with:
- Waters: Waters has developed two kinds of ion mobility (IMS) mass spectrometers: the SELECT SERIES and the SYNAPT XS. Both spectrometers are based on ion mobility mass spectrometry, a type of MS that resolves ions by characteristics other than their mass, such as charge and size. The ions would pass through an electric field where smaller ions with a more open structure would move faster through the chamber than the heavier ions. IM-MS increases the limit of detection for all kinds of ions and allows samples to be processed at much higher speeds than conventional MS.
- Shimadzu: Shimadzu has a series of liquid-chromatography mass spectrometers (LC-MS) available for researchers. Each one uses electrospray ionization (ESI) technology to prevent macromolecules from fragmenting as they are being ionized. Furthermore, their intelligent automation pipeline automates the MS research pipeline, reducing the time and effort needed to optimize MS protocols.
- Hitachi: Hitachi has produced its own kind of MS as well: Thermal Desorption Gas Chromatography MS. The inlet system in TD GC-MS features a tube that contains the sorbent that absorbs the gas. The compounds are then desorbed from the sorbent with a thermal desorber. The system is most useful for the enrichment and identification of volatile and semi-volatile compounds. Alternatively, the Hitachi HM1000A is a benchtop MS that provides measurement rates in less than 10 minutes and allows up to 50 samples to be assayed in 8 hours, achieving high-throughput analyses. The assay is especially useful for measuring a specific kind of metabolite: phthalates.
Explore Mass Spectrometry Leasing with Excedr
Researchers across the R&D sector are using tissue cultures in important preclinical research. Characterizing the biomolecules that comprise these cells is critical for profiling cellular behaviors and developing novel therapeutics for treating disease.
Excedr’s leasing program helps you acquire the exact mass spectrometer you’ll need to advance your proteomics and metabolomics research. Once you know the kinds of biomolecules you’re testing, the ways you’ll process your samples, and the ways you’ll analyze your data, speak with us to start the approval process.