16: LC-MS
Summary
LC-MS is a method for separating mixtures of substances and identifying the components. In biological research, LC-MS is often used to identify the individual proteins present in a mixture.
Also known as
Liquid chromatography-mass spectrometry
Samples needed
In biology, most LC-MS is performed on a solution containing a mixture of proteins
Method
The goal of an LC-MS experiment is to identify as many proteins as possible from a mixture. Often, the proteins are first digested with a protease into a complex mixture of peptides. Then the peptides are subjected to liquid chromatography, a type of column chromatography , to separate the peptides so that only one or a few peptides at a time are eluted into the interface device. The type of column used determines which proteins enter the interface earlier vs. later.
Mass spectrometry is an analytical method in which the mass-to-charge ratio (m/z) of ions can be determined. Therefore, peptides that enter the mass spec must be charged. If this is not already the case, it is achieved by one of several interface methods between the LC and the mass spec, including the popular electrospray ionization. The peptides then pass through a tandem mass spec, where the first device measures the m/z of the intact peptide (parent ion), the peptide is then fragmented, and the second mass spec determines the m/z of each fragment (daughter ions). The resulting spectra, called “peptide fingerprints,” are then computationally compared to a database of theoretical spectra of digest peptides of known proteins. Ultimately, this results in a list of protein “hits,” identifying the proteins that were present in the original mixture before digestion.
Interpretation
This experiment was performed during the course of a project attempting to develop an LC-MS/MS assay for the liver disease biomarker ALT1 in human serum. The top panel in a shows what is called a total ion chromatogram. This means that the y axis measures abundance of any peptides being eluted from the column over the course of the entire run. The middle panel in a is the extracted ion chromatogram. This means that the user has asked the program to show only ions of a particular m/z, in this case, 521.3-522.3. We see that a parent ion with this m/z was eluted between 34 and 35 minutes into the run. Finally, the bottom panel in a shows the MS/MS spectrum containing the daughter ions from the fragmentation of the parent ion. This result is what will be compared to the database. Panel b shows the peptide that makes up the parent ion in this case. In MS/MS, most peptides are fragmented at the peptide bonds, resulting in a series of y and b ions, shown in panel b. Note that the y5 ion (PPLEK) is highlighted in cyan. Panel c shows the results when the daughter ion spectrum from a was submitted to the Mascot tool. The tool identified the analyte from the panel a spectrum as the MTILPPLEK tryptic peptide from ALT1. The most abundant ion in both the theoretical and actual spectra (panels c and a, respectively,) is the y5 ion highlighted in panel b.
Image Descriptions
Figure 1 image description:
Panel a, top: A chromatogram, showing abundance on the y axis and time on the x axis. The chromatogram shows abundant peaks over the entire course of the 60-minute run, especially from 15 minutes onward.
Panel a, middle: A chromatogram, showing a single peak between 34 and 35 minutes. The label says “XIC, m/z = 521.3-522.3.”
Panel a, bottom: A mass spectrum, showing multiple peaks, with the most abundant peak at m/z = 583.46.
Panel b: The chemical structure of the peptide MTILPPLEK. At each peptide bond is a line, with a b ion on the left and a y ion on the right. The b1 ion is the first amino acid, M, and the b8 ion is the first 8 amino acids, MTILPPLE. The y1 ion is the last amino acid, K, and the y8 ion is the last 8 amino acids, TILPPLEK. The y5 ion, PPLEK, is highlighted.
Panel c: An image capture of a web page, Mascot search results. The results show a theoretical mass spectrum that closely matches the real spectrum from panel a. The peptide is identified as MTILPPLEK from ALAT1_HUMAN, alanine aminotransferase 1. ↵
Thumbnail
"Mass spectrum brassicasterol.png" ↗ by Smmudge is licensed under CC BY-SA 3.0 ↗.
Description: Mass spectrum of brassicasterol.
Author
Katherine Mattaini, Tufts University