Mass Spectrometry

For complex proteome analysis Larial offers a variety of peptide mass fingerprint and ion fragmentation sequencing services depending on the types of samples available for analysis. Typical samples may consist of cells or biological tissues/fluids that might require prior fractionation by gel electrophoresis, isoelectric focusing or chromatography to enrich specific subsets of proteins of interest. In gel or solution proteolytic digests are generated to produce primary peptides with ionization potentials suitable for mass spectrometry. LC/MS, LC/MS/MS or MALDI MS is then performed so that peptide identifications can be made using search engines like MASCOT, PROFOUND and MS-FIT .

Our normal MS workflow strategy for in gel digests is to obtain peptide mass fingerprints with sufficient sequence coverage to permit tentative protein identifications. Ion fragmentation sequencing of one or two abundant peptides is then carried out to confirm the protein's identity. This simple paradigm is usually successful if the complexity of the protein sample is reasonably low. If the amount of sample available is limited and/or its complexity great, our approach is usually to perform LC/MS/MS on proteolytic digests to determine the identities of as many proteins as possible. Larial also provides analytical support for a variety of isotopic and tandem mass-tagging technologies that permit relative measurements of protein expression levels in different samples. In these types of studies, peptides in separate digests are chemically-tagged then mixed prior to LC separation and MS analysis. Relative amounts of the proteins in samples can then be deduced from the relative ionization ratios of the differentially tagged peptides observed in MS spectra.

Juxtaposed to the complexity of the cellular proteome is the diversity of structures and chemistries that can exist within a population of purified proteins. The number of variants within a population of purified proteins is best evaluated by MS exact massing. Even the highest purity protein preparations can consist of multiple chemical and conformational variants that can pose difficulties with respect to i) purification, ii) crystallization for structural analysis or iii) regulatory approval as commercial biotherapeutics. Such variants are easily identified by differences between predicted and experimentally observed m/z values in MS spectra. Important in this regard are the potential involvement of side-chain modifications on protein structure/function relationships and the impact of purification protocols and reagents on protein chemistry. Larial has used exact massing of proteins to deduce the authenticity of molecular constructs and has identified important chemical modifications that occur during protein expression and purification. Such information is especially relevant when expressed proteins are destined for further scientific study or more importantly, the commercial biotherapeutics market.