Multivariate analysis of single quadrupole LC-MS spectra for routine characterization and quantification of intact proteins.
Modern high-throughput proteomic platforms allow incomparable protein mixture resolution and identification. However, such sophisticated facilities are expensive and not always accessible for routine analysis of simple mixtures. In this paper, we propose a simple methodology, based on detection of intact, nondigested proteins by LC coupled to single quadrupole MS (sqLC-MS), followed by the analysis of the resulting spectra by multivariate analysis (MA). By doing so, even large molecular weight (MW) proteins, generating complex spectra, can be characterized to a level that allows isoform discrimination, while standard algorithms, such as MS spectrum deconvolution, cannot. To demonstrate the effectiveness of the proposed approach, we have analyzed the spectra of a set of purified, intact albumins from seven different organisms (bovine, human, rabbit, rat, sheep, mouse, and pig) as a model of microheterogenous proteins, using Projection to Latent Structure Discriminant Analysis (PLS-DA). Although these proteins are very similar (less than 1% difference in MW), sqLC-MS/MA allowed their classification, and the identification of unknown source samples. In addition, MA allowed precise protein quantification from the same data (calibration curve R2 = 0.9966). The ability to rapidly characterize and quantify proteins, together with simplicity and affordability, could make of combined sqLC-MS/MA a routine method for the characterization of simple mixture of known proteins.
Michaud
François-Thomas
FT
Département Génie Chimique, Université Laval, Québec, Qc, Canada.
Garnier
Alain
A
Lemieux
Lise
L
Duchesne
Carl
C
D002853:Chromatography, Liquid / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods*; D008970:Molecular Weight; D015999:Multivariate Analysis; D011506:Proteins / Q000032:analysis* / Q000737:chemistry; D040901:Proteomics / Q000379:methods
D011506:Proteins