Question

YOU HAVE DECIDED TO ADD A MASS SPECTROMETER TO YOUR HPLC EQUIPMENT BECAUSE YOU NEED TO SEPARATE AND QUANTIFY SYNTHETIC PEPTIDES IN REACTION VESSELS. THE PEPTIDES WILL BE OF FIVE OR FEWER AMINO ACID RESIDUES. YOU NEED TO DETERMINE THE MOLECULAR WEIGHTS OF THE PEPTIDES ± 3 AMU AND TO ESTIMATE THE RELATIVE AMOUNTS OF EACH PEPTIDE USING ALANINE-TRYPTOPHAN-LEUCINE AS A STANDARD. WHAT TYPE OF MASS SPECTROMETER WILL YOU SELECT TO MEET THE REQUIREMENTS OF THE METHOD? EXPLAIN WHY YOU MADE YOUR SELECTION.

Magnetic sector and Q-IT-TOF are theoretically possible but let’s not take time trying to make something fit that doesn’t. Any TOF mass analyzer could measure masses as low as 150 but lower masses are often difficult to distinguish from background. Magnetic sector mass analyzers can, in principle, be interfaced with an HPLC but this is technically impractical. Ion Trap mass analyzers are generally used in combination with quadrupoles. So the remaining contenders are:

Quadrupole (single, tandem MS)

Quadrupole-Ion Trap (QIT)

Fourier Transform Ion Cyclotron Resonance (FT-ICR)

So select one from the above menu and make a case for your selection. In other words, what features of your selected instrument are important for performing the required measurements?

Answer 1

Ans: The coupling of high-performance liquid chromatography (HPLC) instruments with the right kind of mass spectrometer would be very essential in order to achieve the measurement of the molecular mass of analytes with a high degree of precision and accuracy. The traditional way of achieving correct mass measurements would involve enzymatic cleavage of protein mixture followed by treatment of resulting peptides with various chemicals or physical means so as to achieve their separation. This was followed by mass measurement of respective peptides with mass spectrometry. In this case, it is essential to achieve the separation and quantification of synthetic peptides simultaneously. Additionally, the relative abundance of each peptide present in the reaction vessel has to be measured along with their molecular mass. The accurate quantitation of synthetic peptides usually requires to hydrolyze them partially or completely which is a time-bound process. So as per my perception or thought, it would be essentially achieved through Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FT-ICR). This method is highly sensitive that it can measure up to attomoles (10-18) of peptide concentration. Under the constant magnetic field, the cyclotron frequency of the separated ions is measured. The frequency of charged ions moving in a circular motion and are perpendicular to the direction of the applied magnetic field is termed as cyclotron frequency. And this frequency forms basis for determining the mass to charge ratio or m/z ratio of the charged ions. The separation of charged ions according to relative mass is achieved by this instrument through radio-frequency pulses that were emitted through the electrodes. In this way the method is applied for the efficient quantification of peptides (relative abundance) and their molecular mass.