The ProteoMiner and the FortyNiners: Searching for gold nuggets in the proteomic arena

Abstract

The present review covers modern aspects of combinatorial peptide ligand libraries (CPLL), as used to analyze the ''lowabundance proteome'' in association with mass spectrometry. First, the capturing properties of baits of different lengths (from single amino acid to hexa-peptides) are described to show that a plateau is rapidly reached above a tetra-peptide in length, thus confirming the validity of having adopted hexapeptides for the considered application. The mechanism of interaction with proteins from very complex proteomes and the ability to decrease the dynamic concentration range is demonstrated with the help of mass spectrometry analysis. Examples are given on how treatment with CPLLs dramatically improves the detectability of peptides in mass spectrometry analysis, permitting detection of a very large number of proteins as compared with control, untreated samples. The use of complementary libraries is discussed with the aim to discover additional low-abundance species that escaped the first library. A discussion on the possibility to discover extremely rare gene products, and the quantitative aspect of the technology when associated with mass spectrometry is also provided. Some insights on the applications for hidden, low-abundance biomarkers are also presented. #

Comprehensive Characterization of Extracellular Herpes Simplex Virus Type 1 Virions

“…It should be pointed out that quantitative mass spectrometry of complex protein mixtures is still being actively pursued by mass spectrometry specialists (2,73,82,85). At issue is the different behavior of different proteins and peptides during separation, ionization, and solvation, not including relative abundance and potential unpredictable posttranslational modifications.…”

Characterization of the bovine milk proteome in early-lactation Holstein and Jersey breeds of dairy cows

“…The low-abundance minor proteins were enriched by the ProteoMiner Kit (BioRad Laboratories, Hercules, CA, USA) and 32 mg of whey protein was added to 100 μl of ProteoMiner beads. The whey samples were gently shaken with individual ProteoMiner columns for 2 h at room temperature and columns were washed thoroughly using HPLC grade water to remove excess proteins [37, 38]. The low abundance proteins were eluted off the beads by addition of 20 μl 4x Laemmli sample buffer (8% SDS, 40% glycerol, 250 mM Tris, pH 6.8, 400 mM DTT with trace amount of bromophenol blue).…”

Plasma proteomics for biomarker discovery: A study in blue

“…Yet, at least two methodologies, devised for digging deeper into the low-abundance proteome, have gone unscathed so far: immuno-depletion of the 7, 14 or 20 most abundant proteins in biological fluids [6] and combinatorial peptide ligand libraries (CPLLs, as reviewed in, e.g. [7][8][9]). In the first case, the major problem, together with accidental co-depletion of untargeted species [10], is the much too low volume of plasma that can be handled, typically 100 mL.…”