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Mass Spectrometry

Introduction to Mass Spectrometry

Mass spectrometry (mass spec or MS), is an important analytical tool to measure molecular mass and is used in industry and academia for both routine and research. MS is the principle methodology in proteomic research and is used to determine the quantification, characterization and structural information of proteins. MS involves separating mixtures of substances into single components by ionization.

The two most widely used methods for ionization of proteins is matrix-assisted laser desorption / ionization (MALDI) and electrospray ionization (ESI). Most ionization methods result in creation of both positively and negatively charged sample ions and is dependent on proton affinity of the sample. The relative amounts of charged ions should be decided prior to analysis.

A mass spectrometer consists of three fundamental parts (see Figure 1): the ionization source, the analyzer and the detector.  Both the analyzer and the detector, and often the ionization source, are maintained under high vacuum to help facilitate ion travel from one end of the instrument to the other. This also prevents interference from molecules of air. The data system in modern MS instruments controls the entire operation from ionization to detection, and often the sample introduction process too, depending on the ionization process used.

 

                  Figure 1: Components of a Mass Spectrometer

Ionization Source

The sample must first be introduced into the ionization source (e.g., MALDI / ESI). Here, the molecules (particles) within the sample are ionized and become easier to manipulate compared with neutral molecules.

Analyzer

The ionized particles are directed into the analyzer section of the mass spectrometer. Here they are separated / resolved according to their mass (m) to charge (z) ratios (m/z). The more commonly known analyzers include quadrupoles, time of flight (TOF), magnetic sectors, and both Fourier transform (FT) and quadrupole ion traps. Each have different features, which should be carefully considered in conjunction with the ionization source.

Detector

The most common detectors used in MS are the photomultiplier and electron multiplier. Separated ions move into the detector where their signal is detected, monitored and amplified. Information regarding their m/z ratios and relative abundance are transmitted to the data system, where it is recorded as mass spectra.

The m/z ratios are subsequently plotted versus their intensities. This interprets the number and molecular mass for each component of the sample, along with relative abundance. The resulting stored data is presented in the form of a m/z spectrum.

Some advantages and applications of MS are given below:

Advantages :

  • High sensitivity
  • High mass accuracy
  • Ability to monitor reactions, sequence amino acids, and oligonucleotides
  • Provides structural information
  • Identify and quantify samples

Applications:

  • Shotgun mass spectrometry
  • Protein identification
  • Quantitative proteomics
  • Peptide sequencing
  • Identification of post translational modifications

Protein Characterization

There are two main approaches for MS protein characterization.

Bottom Up Proteomics

In the conventional MS procedure referred to as the “bottom up” approach, the protein sample is enzymatically digested (using proteases such as trypsin or pepsin) or chemically (using other proteolytic agents), into smaller peptides either in solution or in gel, following either electrophoretic or chromatographic separation. The resultant selection of peptide products is then introduced to the mass analyzer.

The term peptide mass fingerprinting (PMF) is given to the method for identifying a protein based on the characteristic pattern of peptides. However, when the method used for identification of the protein is performed using sequence data determined from tandem MS (MS-MS) analysis, it is termed de novo peptide sequencing. Tandem (MS-MS) mass spectrometers are those with more than one analyzer, which do not have to be of the same type and are useful for structural and sequencing studies.

Nevertheless, the bottom up approach has inherent limitations. For example, only a small and variable fraction of the total peptide population of a protein can be recovered, resulting in low / limited percentage coverage of the protein sequence. This means that much of the information about location of post translational modifications (PTMs), sequence variants such as mutants and different protein isoforms are lost.

Top Down Proteomics

In the method referred to as “top down”, intact proteins are converted to their ionized form, using one of the techniques already described above (e.g., MALDI / ESI), followed by introduction to a mass analyzer. Labile structural protein characteristics, which are mostly destroyed in the bottom up method, remain preserved and all PTMs can be analysed concurrently in one spectrum. This approach is generally limited to low throughput single protein studies.

The top down method can overcome the information loss problems seen with the bottom up approach, as it can measure the intact protein’s isoforms – provided they have sufficiently different masses. Sample preparation is simplified and time consuming protein digestion, required for the bottom up method, is eliminated. Top down has certain advantages over bottom up but faces technical challenges before it becomes a robust method for proteomic research. This in part is due to the complexity of protein handling, meaning that the upper limit for proteins is less than 50 kDa.

 

A third approach currently in development sees a hybrid form of bottom up and top down methods. An intermediate “middle-down” approach involves limited proteolytic digestion of larger native proteins into more manageable polypeptides, followed by the top down method. This results in better sequence analysis and retained PTM information.

Sample preparation

Successful sample preparation is a key step during any analytical procedure and begins with a defined experimental design. Important steps in sample preparation include proteolytic digestion of proteins into peptide fragments, and peptide fractionation prior to MS analysis.

Expedeon offers a range of products that can assist you in sample preparation ready for MS (see Figure 2).

 

Expedeon Products used in Sample Preparation for Mass Spectrometry

Figure 2: Range of Expedeon products for Mass Spectrometry

 

FASP Protein Digestion Kit

Expedeon’s Filter Aided Sample Prep (FASP) Protein Digestion Kit provides complete and efficient protein solubilization and trypsin digestion of samples for proteome analysis, even in the presence of extreme contaminants. The kit facilitates highly efficient extraction or digestion. FASP is also the enabling technology behind quantitative MS analysis of FFPE archived tissues. The resulting filtrate is free of detergents, large molecules, and other substances that would interfere with MS analysis of the proteome.

 

GELFrEE 8100 Fractionation System

Expedeon’s GELFrEE 8100 Fractionation System and Cartridge Kits for protein fractionation, partitions complex protein mixtures over a wide range into enriched user selectable liquid phase molecular weight fractions. Each of the GELFrEE 8100 Fractionation Cartridge Kits contain eight channels for molecular fractionation and liquid phase recovery. The eight independent GELFrEE 8100 Cartridge channels consist of a precision cast gel column surrounded by pipette accessible sample loading and fraction collection chambers. Specialized materials optimize protein recovery and prevent nonspecific loss. A dynamic range (up to 500kDa) of complex protein mixtures are simplified and reduced for bottom up discovery proteomics, and intact proteins are fractionated and recovered for top down proteomics.

 

PPS Silent® Surfactant

Expedeon’s PPS Silent® Surfactant disrupts cell membranes, solubilizes hydrophobic proteins and improves efficiency of enzymatic (trypsin) digestion. PPS also enables membrane proteomic analysis of cells captured by light confocal microscopy (LCM), and isobaric tag for relative and absolute quantitation (iTRAQ) of insoluble proteins Unlike first generation cleavable surfactants for MS, at low (acidic) pH PPS is rapidly hydrolyzed into soluble, nonsurfactant cleavage products with no detergent interference, meaning that there is no oily film or sticky pellet to complicate sample preparation.

 

PolyMAC

Polymer based Metal ion Affinity Capture (PolyMAC), overcomes the shortcomings of current enrichment methods that typically lack required reproducibility, phosphopeptide recovery and selectivity. PolyMAC offers an efficient and greatly improved method to achieve more complete phosphopeptide enrichment under homogenous conditions. In this method, a soluble nanopolymer is functionalized with metal ions to specifically capture O-phosphorylated peptides. Expedeon offers both a magnetic bead based purification method and a spin column based method.

 

References:

Ashcroft, AE. An Introduction to Mass Spectrometry. Available at: http://www.cerm.unifi.it/static/piccioli/MS1.pdf (Accessed September 2018).

Chait, BT. Mass Spectrometry: Bottom-Up or Top-Down? Science. 2006;314:65–66.

Chait, BT. Mass spectrometry in the postgenomic era. Ann Rev Biochem. 2011;80:239–46.

Dalmasso, E., et al. Top-down, Bottom-up – The Merging of Two High-Performance technologies. Available at: https://pdfs.semanticscholar.org/6072/4fd17b02cd71397dc4f7800358b7d484482c.pdf (Accessed September 2018).

Ge Y, et al. Top down characterization of larger proteins (45 kDa) by electron capture dissociation mass spectrometry. J Am Chem Soc. 2002;124:672–678.

Wheir, T. Top-Down versus Bottom-Up Approaches in Proteomics. LC-GC chromatographyonline.com 2006: Issue 9. Available at: http://www.chromatographyonline.com/top-down-versus-bottom-approaches-proteomics-0 (Accessed September 2018).

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