MS-based methods for identifying post-translational modification gradually took over other proteomic approaches because of its sensitivity of detection, low run time and reduced biochemical biasness (this is in reference to specific dyes used to stain specific modifications). The use of mass spectrometry based PTM analysis goes way back to the time when scientists characterized the spectrum of hemoglobin from sickle cell anemia. Sickle cell anemia results from a change in one nucleotide of a codon resulting in the change in one amino acid, from glutamic acid to valine. This change in the hemoglobin is evident not only on the phenotype but also on the mass spectrum as well. The fragmentation pattern of both normal and sickle cell hemoglobin are the same. However, one of the ion peaks shifts showed change in mass of 30 Da in case of sickle cell anemia.
Every post-translational modification adds a specific mass to the protein, depending on the number of modifications. For example, a single phosphorylation in the protein would increase the overall protein mass by a magnitude of 80 Da. Identifying the amount of change in the spectrum shift is thus a direct measure of the post-translational modification, in terms of both the number of modifications as well as the site of modification (using tandem MS/MS splitting and sequence determination). However, like every other techniques, MS based approach also faces several limitations, which would be discussed.

Post- translational modifications can be detected by means of mass spectrometry due to the unique fragmentation patterns of phosphorylated seine and threonine residues.. The modified protein of interest is digested into smaller peptide fragments using a suitable enzyme like trypsin. This digest is then mixed with a suitable organic matrix such as a-cyano-4-hydroxycinnamic acid, sinapinic acid etc. and then spotted on to a MALDI plate.
The target plate containing the spotted matrix and analyte is placed in a vacuum chamber with high voltage and short laser pulses are applied. The laser energy gets absorbed by the matrix and is transferred to the analyte molecules, which undergo rapid sublimation resulting in gas phase ions. These ions are accelerated and travel through the flight tube at different rates. The lighter ions move rapidly and reach the detector first while the heavier ions migrate slowly. The ions are resolved and detected on the basis of their m/z ratios and a mass spectrum is generated.
Identification of PTMs by MS largely lies in the interpretation of results. Comparison of the list of observed peptide masses from the spectrum generated with the expected peptide masses enables identification of those peptide fragments that contain any PTM due to the added mass of a modifying group. In this hypothetical example, two peptide fragments are found to have different m/z values, differing by 80 daltons and 160 daltons. It is known that the added mass of a phosphate group causes an increase in m/z of 80 daltons. Therefore, this principle of mass difference enables detection of modified fragments.

Illustration: Methodology for MALDI analysis