A novel fractionation method prior to MS-based proteomics analysis using cascade biomimetic affinity chromatography

Staged-probability strategy of processing shotgun proteomic data to discover more functionally important proteins

“…Using peptides with a probability (p) value greater than or equal to 95% as correct filtering conditions, the original LC/ MS datasets of rat-liver proteomics by Tan et al (Tan et al, 2009) identified 391 non-redundant proteins in unfractionated rat liver cytosol, 499 proteins in 2 fractions of the first layer affinity fractionation, 616 in 4 fractions of L2 fractionation, and 738 in 8 fractions of L3 fractionation.…”

“…Original LC/MS datasets of rat-liver proteomics were collected using cascade affinity fractionation and Trans-Proteomic Pipeline (TPP) software (Tan et al, 2009). Briefly, the cascade affinity fractionation was composed of different biomimetic affinity ligand columns with a diverse combination of characteristics for affinity fractionation at cascade level.…”

“…MS results of all sample fractions from each one layer were combined. Tan et al found that the number of proteins identified increased as more fractionation layers were used (Tan et al, 2009).…”

“…In this study, the biological meaning of probability was rediscovered. We propose a novel staged-probability approach (SPA) for the assessment of shotgun proteomic data in order to identify more functionally important proteins, exemplified by LC/MS datasets of rat liver proteins pretreated by cascade affinity chromatography and processed by the Trans-Proteomic Pipeline (TPP) software series (Tan et al, 2009).…”

The Improvement of LC-MS/MS Proteomic Detection with Biomimetic Affinity Fractionation

“…To simplify the sample and increase the detection probability of middle-and low-abundance proteins, suitable high abundance protein depletion and prefractionation methods were often adopted prior to trypsin digestion (Bergh et al, 2003;Jacobs et al, 2004;Gao et al, 2006;Liu and Zhang, 2007;Gao et al, 2008). Affinity chromatography (AC) can be also used to reduce the complexity of the protein/peptide by selecting the specific target protein/peptide (Gygi et al, 1999;Pieper et al, 2003;Greenough et al, 2004, Tan et al,2009a,2009b& 2010. Traditional affinity adsorbents comprise natural biological ligands, which are expensive to produce, show low binding capacities, have limited life cycles, and low scale-up potential.…”