A comparison of immobilized pH gradient isoelectric focusing and strong-cation-exchange chromatography as a first dimension in shotgun proteomics.
Recently, we have developed a high-resolution two-dimensional separation strategy for the analysis of complex peptide mixtures. This methodology employs isoelectric focusing of peptides on immobilized pH gradient (IPG) gels in the first dimension, followed by reversed-phase chromatography in the second dimension, and subsequent tandem mass spectrometry analysis. The traditional approach to this mixture problem employs strong-cation-exchange (SCX) chromatography in the first dimension. Here, we present a direct comparison of these two first-dimensional techniques using complex protein samples derived from the testis of Rattus norvegicus. It was found that the use of immobilized pH gradients (narrow range pH 3.5-4.5) for peptide separation in the first dimension yielded 13% more protein identifications than the optimized off-line SCX approach (employing the entire pI range of the sample). In addition, the IPG technique allows for a much more efficient use on mass spectrometer analysis time. Separation of a tryptic digest derived from a rat testis sample on a narrow range pH gradient (over the 3.5-4.5 pH range) yielded 7626 and 2750 peptides and proteins, respectively. Peptide and protein identification was performed with high confidence using SEQUEST in combination with a data filtering program employing pI and statistical based functions to remove false-positives from the data.
Essader
Amal S
AS
Research Triangle Institute, Research Triangle Park, NC 27709-2194, USA.
Cargile
Benjamin J
BJ
Bundy
Jonathan L
JL
Stephenson
James L
JL
D000818:Animals; D002852:Chromatography, Ion Exchange; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing / Q000379:methods; D008297:Male; D013058:Mass Spectrometry; D010455:Peptides / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*; D040901:Proteomics; D051381:Rats; D013737:Testis / Q000737:chemistry*
D010455:Peptides; D011506:Proteins