Abstract
Novel approaches for the qualitative and quantitative proteomics analysis by nanoscale LC-MS applied to the study of protein expression response in depleted and undepleted serum of Gaucher patients undergoing enzyme replacement therapy are presented. Particular emphasis is given to the method reproducibility of these LC-MS experiments without the use of isotopic labels. The level of chitotriosidase, an established Gaucher biomarker, was assessed by means of an absolute concentration determination technique for alternate scanning LC-MS generated data. Disease associated proteins, including fibrinogens, complement cascade proteins, and members of the high density lipoprotein serum content, were recognized by various clustering methods and sorting and intensity profile grouping of identified peptides. Condition-unique LC-MS protein signatures could be generated utilizing the measured serum protein concentrations and are presented for all investigated conditions. The clustering results of the study were also used as input for gene ontology searches to determine the correlation between the molecular functions of the identified peptides and proteins.
Highlights
Novel approaches for the qualitative and quantitative proteomics analysis by nanoscale LC-MS applied to the study of protein expression response in depleted and undepleted serum of Gaucher patients undergoing enzyme replacement therapy are presented
LC-MS-based relative quantification methods have emerged to identify and quantify peptides and proteins in mixtures of various complexities. The majority of these relative quantification techniques use the introduction of stable isotopes into the samples including ICAT [10], isobaric tag for relative and absolute quantification [11], in vivo stable isotope labeling by amino acids in cell culture (SILAC) [12], and 18O labeling [13, 14]
A recent independent study from the Association of Biomolecular Resource Facilities evaluated quantitative proteomics approaches, and it was concluded that label-free methods did at least as well as stable isotope labeling methods
Summary
Label-free LC-MS quantification methods have been described to determine relative abundances of proteins between multiple conditions (18 –24). These methods are typically based on determining peak area ratios of the same peptides between different conditions. Silva et al [25] discovered that a label-free approach allows for the estimation of absolute protein concentrations, which were subsequently used for stoichiometry studies. The Gaucher disease protein serum profile was examined as it is biochemically and quantitatively well defined. Intensity profiling by K-means clustering of identified peptides was used to identify interrelating proteins, for example proteins that are components of the same biochemical pathway
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