Mass Spectrometry Detects Covalent Modifications

Mass spectrometry, which discriminates molecules based solely on their mass, is ideal for detecting the phosphate, hydroxyl, and other groups on posttransla-tionally modified amino acids. Each adds a specific and readily identified increment of mass to the modified amino acid (Table 4-2). For analysis by mass spectrometry, a sample in a vacuum is vaporized under conditions where protonation can occur, imparting positive charge. An electrical field then propels the cations through a magnetic field which deflects them at a right angle to their original direction of flight and focuses them onto a detector (Figure 4-8). The magnetic force required to deflect the path of each ionic species onto the detector, measured as the current applied to the electromagnet, is recorded. For ions of identical net charge, this force is proportionate to their mass. In a time-of-flight mass spectrometer, a briefly applied electric field accelerates the ions towards a detector that records the time at which each ion arrives. For molecules of identical charge, the velocity to which they are accelerated—and hence the time required to reach the detector—will be inversely proportionate to their mass.

Conventional mass spectrometers generally are used to determine the masses of molecules of 1000 Da or less, whereas time-of-flight mass spectrometers are suited for determining the large masses of proteins. The analysis of peptides and proteins by mass spec-tometry initially was hindered by difficulties in volatilizing large organic molecules. However, matrixassisted laser-desorption (MALDI) and electrospray dispersion (eg, nanospray) permit the masses of even large polypeptides (> 100,000 Da) to be determined with extraordinary accuracy (± 1 Da). Using electro-spray dispersion, peptides eluting from a reversed-

Table 4-2. Mass increases resulting from common posttranslational modifications.

Modification

Mass Increase (Da)

Phosphorylation

80

Hydroxylation

16

Methylation

14

Acetylation

42

Myristylation

210

Palmitoylation

238

Glycosylation

162

Figure 4-8. Basic components of a simple mass spectrometer. A mixture of molecules is vaporized in an ionized state in the sample chamber S. These molecules are then accelerated down the flight tube by an electrical potential applied to accelerator grid A. An adjustable electromagnet, E, applies a magnetic field that deflects the flight of the individual ions until they strike the detector, D. The greater the mass of the ion, the higher the magnetic field required to focus it onto the detector.

Figure 4-8. Basic components of a simple mass spectrometer. A mixture of molecules is vaporized in an ionized state in the sample chamber S. These molecules are then accelerated down the flight tube by an electrical potential applied to accelerator grid A. An adjustable electromagnet, E, applies a magnetic field that deflects the flight of the individual ions until they strike the detector, D. The greater the mass of the ion, the higher the magnetic field required to focus it onto the detector.

phase HPLC column are introduced directly into the mass spectrometer for immediate determination of their masses.

Peptides inside the mass spectrometer are broken down into smaller units by collisions with neutral helium atoms (collision-induced dissociation), and the masses of the individual fragments are determined. Since peptide bonds are much more labile than carboncarbon bonds, the most abundant fragments will differ from one another by units equivalent to one or two amino acids. Since—with the exception of leucine and isoleucine—the molecular mass of each amino acid is unique, the sequence of the peptide can be reconstructed from the masses of its fragments.

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