Tagless extraction-retentate chromatography: a new global protein digestion strategy for monitoring differential protein expression.
A new global protein digestion and selective peptide extraction strategy for the purpose of monitoring differential protein expression, coined as tagless extraction-retentate chromatography, is introduced. Target protein populations are firstly digested under reduced and alkylated conditions, and resultant peptides selectively extracted via covalent attachment to methionine residues by bromoacetyl reactive groups tethered to the surface of glass beads packed in small reaction vessels. After conjugation, reactive beads are stringently washed to remove nonspecifically bound peptides and then later treated with beta-mercaptoethanol to release captured methionine peptides in their nascent state, without complicating affinity tags. Recovered methionine containing peptides are profiled using the surface-enhanced laser desorption/ionization (SELDI) retentate chromatography mass spectrometry (RCMS) method. Selected peptides are further studied employing ProteinChip tandem mass spectrometry (MS/MS) analysis to identify their parent proteins. This approach has been applied to an Escherichia coli lysate model system and has demonstrated facility in reducing global digest complexity, sensitivity to low protein expression levels, and significant quantitative capability. It is envisioned that tagless extraction-RCMS will evolve to be a valuable approach for both basic research and clinical proteomics endeavors.
Weinberger
Scot R
SR
Ciphergen Biosystems, Inc., Fremont, CA 94555, USA. sweinberger@ciphergen.com
Viner
Rosa I
RI
Ho
Patrick
P
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002845:Chromatography / Q000379:methods* / Q000706:statistics & numerical data; D029968:Escherichia coli Proteins / Q000235:genetics / Q000302:isolation & purification; D013058:Mass Spectrometry / Q000379:methods* / Q000706:statistics & numerical data*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000302:isolation & purification; D040081:Protein Array Analysis / Q000379:methods* / Q000706:statistics & numerical data; D011506:Proteins / Q000302:isolation & purification*; D040901:Proteomics / Q000379:methods / Q000706:statistics & numerical data; D012680:Sensitivity and Specificity; D012710:Serum Albumin, Bovine / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods / Q000706:statistics & numerical data
D029968:Escherichia coli Proteins; D010446:Peptide Fragments; D011506:Proteins; D012710:Serum Albumin, Bovine