Other local proteomic services that may be of interest:
Mass Spectrometry Measurements for Proteomics
- Verify known protein
- Determine type and amount of post-translational modification to known protein
- First pass analysis for unknown protein
- Verify known protein by matching to database entries
- Identify unknown proteins by matching to database entries
- Localize post-translational modifications to individual peptides
- Identify unknown proteins using partial sequence and database matching
- Localize post-translational modifications to individual amino acids
- Verify known proteins
ESI mass spectrometry (MS) was first published by Yamashita and Fenn1 in 1984, but it was based on earlier work by Dole, Iribarne, and Thompson. The advent of nanoelectrospray in 1996, published by Wilm and Mann, increased the use of ESI MS, particularly when samples were limited in concentration or volume. Two theories about the mechanism of ESI have been proposed: the charged residue model and the ion ejection model. In the charged residue model, the total charge of the droplet remains the same as the solvent evaporates, and it is left on the non-volatile components. In the ion ejection model, the solvent evaporates from the droplets until the force from Coulombic repulsion overcomes the surface tension of the droplet and individual ions are expelled. In either case, the resulting ions are highly charged, and each has a distribution in the number of charges (called the charge state distribution). For biological molecules such as peptides and proteins, the charge is introduced by protonation or deprotonation.
Preparation of Samples
The analyte of interest is usually dissolved in aqueous solution and mixed with a similar volume of volatile organic solvent. This solution is then loaded into the nanospray emitter for single sample analysis or it can be subjected to liquid chromatography before it is electrosprayed (LC-ESI MS).
Analysis of Samples
Each sample is sprayed using a voltage difference between the ESI needle and the orifice plate. In certain cases, backpressure or sheath gases are used to aid in nebulization of the spray. In this instrument, the electrospray ionization is performed at atmospheric pressure. Then, the ions are introduced into the vacuum using several directing optics, while the solvent and other neutral molecules are excluded using skimmers. In this particular instrument, the ions are transported and focused through quadrupoles (denoted as the primary beam); then, mass analysis is done using an orthogonal time-of-flight (TOF) mass spectrometer. Tandem mass spectrometry (MS/MS) can also be performed in the primary beam using collision-induced dissociation (CID), so this instrument is configured primarily for peptide sequencing.
- Yamashita, M. and Fenn, J.B. (1984). Electrospray ion source. Another variation on the free-jet theme. J. Phys. Chem. 88, 4451-4459.
MALDI MS was first published by Michael Karas and Franz Hillenkamp1 in 1985 when they used tryptophan as a matrix for other amino acids in laser desorption MS experiments. Koichi Tanaka’s group accidentally discovered that mixing sample with metal particles allows the analysis of large molecules such as intact protein. In 1988, both groups published the application of MALDI MS on intact protein using fine metal granules2 and organic acids3 as matrices, respectively.
Preparation of Samples
The analyte of interest is dissolved (usually in aqueous solution) and mixed with a large excess of matrix (1000:1 to 10000:1). The matrix usually an organic acid, such as derivatives of benzoic acid or cinnamic acid, but other compounds also work. A small aliquot of this mixture (0.5 - 1.0 microliter) is deposited on the stainless steel sample stage and allowed to air-dry. For calibration, an internal standard may be spiked into the matrix solution or another spot may be placed nearby for external calibration.
Analysis of Samples
After the sample deposits have dried, the plate is placed in the instrument via a vacuum interlock chamber. Each sample is irradiated with a pulsed nitrogen laser (lambda = 337 nm) to create ions in the gas phase (desorption/ionization). The mass-to-charge ratios (m/z) are measured using an axial TOF mass spectrometer. The ions can be detected in linear mode (higher sensitivity, but lower resolution) or in reflected mode (lower sensitivity, but higher resolution).
- Karas, M., Bachmann, D., and Hillenkamp, F. (1985). Influence of the Wavelength in High-Irradiance Ultraviolet Laser Desorption Mass Spectrometry of Organic Molecules. Anal. Chem. 57, 2935-2939.
- Tanaka, K., Waki, H., Ido, Y., Akita, S., Yoshida, Y., & Yoshida T. (1988). Protein and polymer analysis up to m/z 100,000 by laser desorption time-of-flight mass spectrometry. Rapid Commun. Mass Spectrom. 2, 151-153.
- Karas, M., Hillenkamp, F. (1988). Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons. Anal Chem. 60, 2299-2301.
The ions are rapidly accelerated in the ion source using a high voltage (20 - 25 kV). After this initial acceleration, the ions drift in a field free region until they collide with a detector.
Ep = q*V
- The potential energy of a charged particle in an electric field is determined by the charge on that particle (q) multiplied by the magnitude of the voltage (V). This potential energy is converted into kinetic energy.
- The kinetic energy of particle is equal to the mass of the particle (m) multiplied by the square of the velocity (v) divided by 2. By setting the potential energy and kinetic energy equal, the "time-of-flight equation" can be simply shown: Ep = Ek
qV = ½mv2
- Then, the velocity is replaced by distance divided by time (d/t).
qV = ½m(d/t)2
2V = md2/qt2
(2Vt2)/(d2) = m/q = m/z
- Thus, the mass-to-charge ratio of each ion can be measured.
Spectra are acquired by transient recording. The amount of signal from the detector is recorded over time after each laser shot. The final mass spectrum is presented as an average of the individual time recordings. In these spectra, it is important to consider the signal-to-noise ratio (S/N) of the peaks. Ion signals with poor S/N values may not be reliable; on the other hand, it is important to prevent detector saturation, which will occur if the S/N values of lower mass ion signals are too high. Also, the S/N values must be carefully monitored for isotope ratio measurements.