The fundamental idea behind mass spectrometry as a technique is that you can sep

Question

The fundamental idea behind mass spectrometry as a technique is that you can separatemolecular species based on minute differences in mass/charge ratio. We didn’t talk about itmuch in class, but tandem mass spectrometry (or MS/MS) is the technique coupling onemass spectrometric sorting to another.

a. One of the main purposes for doing tandem mass spectrometry is to gain informationabout the specific sequence of amino acids in a polypeptide. This is useful for identifyingproteins within a band on a gel, or in a mixture. Suppose you run tandem massspectrometry on a protein and determine that the following peptide fragments all comefrom a single polypeptide that was originally 8 to 10 residues long (arranged inincreasing size): SerMet; MetAlaGly; GluThrIle; AlaGlyAsnGlu; GluThrIleSerMet. What was the original sequence of the polypeptide?

b. Why is tandem mass spectrometry, rather than regular mass spectrometry, able to giveinsight into a specific sequence?

c. One of the other main uses for tandem mass spectrometry is identifying precisely whichamino acid within a sequence contains a particular posttranslational modification.Suppose you knew that a protein was phosphorylated on one of the serines in thepolypeptide “SANSASTARK”. How would tandem mass spectrometry allow you todetermine which serine contained the phosphorylation?

Explanation / Answer

Answer:

a. Arranging the peptide fragments in overlapping fashion gives the sequence of the polypeptide:

SerMet

      MetAlaGly            

            AlaGlyAsnGlu

                               GluThrIle

                               GluThrIleSerMet

So, sequence of the polypeptide: Ser-Met-Ala-Gly-Asn-Glu-Thr-Ile

b. In order to acquire structural or peptide sequence information, it is necessary to induce fragmentation of the peptides of interest. This is not possible with soft ionisation techniques such as ESI and MALDI.

However, the use of these techniques in conjunction with tandem mass spectrometry (MS/MS) has allowed the structural elucidation of a wide range of peptides. In MS/MS peptides are individually ionised in the source region using ESI or MALDI. These peptides are then further separated, based on their m/z ratio.

The tandem mass spectrometry data can be used to elucidate the primary sequence of a peptide. The process of deducing an amino acid sequence from the tandem mass spectra is aided by the ready availability of protein and DNA databases.

c. Identification of post-translational modifications (PTMs) involves digesting the proteins into peptides, analyzing the peptides using tandem mass spectrometry (LC-MS/MS), and then searching for modifications using search engines. Most commonly used search engines are SEQUEST or other specialized PTM softwares.

Phosphorylation site determination through tandem mass spectrometry is very much dependent on the particular peptide sequence. A good strategy is to compare the spectra of phosphorylated peptides before and after treatment with phosphatase. Peptides which contained phosphoryl groups will show a decrease in molecular weight. The repeated spectra of different fragments can be compared to know the site of phosphorylation.

The exact serine which was phopsphorylated can thus be accurately determined through the above method.

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