Mass Spectrometry

Mass spectrometry (MS) is an analytical technique for the determination of the elemental composition of a sample or molecule. It is also used for elucidating the chemical structures of molecules, such as peptides and other chemical compounds. The MS principle consists of ionizing chemical compounds to generate charged molecules or molecule fragments and measurement of their mass-to-charge ratios. In a typical MS procedure, a sample is loaded onto the MS instrument. The components are ionized by one of a variety of methods (electrospray, MALDI, or chemical ionization). The ions are directed by an electric and/or magnetic field. Computation of the mass-to-charge ratio (m/z) of the particles is based on the details of motion of the ions as they transit through electromagnetic fields.
MS instruments consist of three modules: an ion source, which can convert gas phase sample molecules into ions (or, in the case of electrospray ionization, move ions that exist in solution into the gas phase); a mass analyzer, which sorts the ions by their masses by applying electromagnetic fields; and a detector, which measures the value of an indicator quantity and thus provides data for calculating the abundances of each ion present. The technique has both qualitative and quantitative uses. These include identifying unknown compounds, determining the isotopic composition of elements in a molecule, and determining the structure of a compound by observing its fragmentation.

Peptide Mapping

Peptide mapping of a protein or oligopeptide can be used for the purposes of identity or release. For example, in developing a trypsin based peptide map, the protein is contained in a volatile buffer solution such ammonium bicarbonate (100 mM) and treated with the enzyme at various enzyme:substrate ratios over a period of time (i.e. 5 to 24 h, typically). Other enzymes that have had applicability are Lys-C, Arg-N, Glu-C, and chymotrypsin. The digested mixture is then separated on a reverse phase U/HPLC system as depending on method requirements.

For each project, digest conditions are examined through reproducibility of the determined method of digestion and provide its impact on sequence coverage percentage (>95% targeted). MS analysis (LC-TOF or LC-MS/MS) is used to determine the sequence identities of key peptide fragments. We will examine the matrix for interferences.

• Sample required: microgram to milligram amounts of protein

N, C-Terminal Sequencing

MS/MS analysis on the LTQ-Orbitrap is performed to determine the N-terminal sequence identities of key peptide fragments. In cases where the sequence is known, a suitable protease will be used to cleave the N-terminal amino acids (~15-25 residues). Sequencing will be performed on the LTQ-Orbitrap. For the determination of N-terminal sequence, additional protease digests (peptide maps) would be required to show overlap with initial protease cleavage site. Manual alignment of the peptides would then be possible.

• Sample required: microgram to milligram amounts of protein

MS Oxidation Assay

A method for the determination of oxidation extent of a protein has been employed. The method examines the LC chromatogram of a protease digested sample with UV/MS-TOF detection. A direct comparison of the oxidized peptide with the native peptide is examined. The native peptide is identified by either MS confirmation or through authentic material if needed. Alternately, the oxidized peptide is identified in the chromatogram and analyzed by LC-MS/MS (MRM) on a triple quadrapole mass spectrometer.

• Sample required: microgram to milligram amounts of protein

Carbamylation

Carbamylation is the addition of urea to a lysine residue (+44 Da). This chemical reaction occurs in development of the products isolation and purification process. To determine the location and extent of the modification, a peptide map is performed on the LTQ-Orbitrap, TSQ Vantage Triple Quad or Agilent TOF. Digest conditions are examined through reproducibility of the determined method of digestion and provide its impact on carbamylation and other possible modifications to the protein. MS/MS analysis is used to determine the sequence identities of key peptide fragments. Matrix interferences (i.e. aluminum content) are also examined.

• Sample required: microgram to milligram amounts of protein

Deamidation

Deamidation is the hydrolysis the asparagine and glutamine side chain amides, of a peptide or protein, releasing ammonia. Overall, the mass difference of on deamidation is 1 Da. This chemical reaction occurs through direct aging of the protein in appropriate pH conditions and possibly in the development of the purification process. To determine the location and extent of the modification, a peptide map is performed. Digest conditions are examined through reproducibility of the determined method of digestion and provide its impact on carbamylation and other possible modifications to the protein. MS/MS analysis is used to determine the sequence identities of key peptide fragments. Matrix interferences (i.e. aluminum content) are also examined.

• Sample required: microgram to milligram amounts of protein