Mass spectrometric identification of serum peptides employing derivatized poly(glycidyl methacrylate/divinyl benzene) particles and mu-HPLC.
Biomarkers play a key role in preclinical screening and diagnosis of a disease. Various support materials are utilized for this task, in combination with MALDI-TOF-MS. The way to effectively bind serum contents and their profiling is well-elaborated by the material-enhanced laser desorption ionization (MELDI) approach. In this particular work, focus is placed on the development of a strategy to identify low molecular weight serum peptides. Poly(GMA/DVB) is derivatized in a way to achieve an affinity termed as immobilized metal ion affinity chromatography (IMAC). Iminodiacetic acid (IDA) is used as a chelating ligand, whereas copper (Cu2+) acts as a metal ion for complexing peptides and proteins out of blood serum. Polymer binds the serum compounds over a broad mass range, which includes low mass peptides and high mass albumin (66 kDa). Bound contents are eluted from material by an acetonitrile/trifluoroacetic acid mixture, which proves the reversible nature of metal and amino acid linkage. Polystyrene/divinyl benzene (PS/DVB) monolithic capillary column is used for fractionation through RP-HPLC, prior to the target spotting. The tandem TOF fragment ion mass spectra of each fraction is acquired and used to search against the Swiss-Prot database, using the Mascot search engine for the identification of peptides.
Rainer
Matthias
M
Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens University, Innrain 52a, 6020 Innsbruck, Austria.
Najam-ul-Haq
Muhammad
M
Bakry
Rania
R
Huck
Christian W
CW
Bonn
Günther K
GK
D000595:Amino Acid Sequence; D015415:Biomarkers / Q000737:chemistry; D001798:Blood Proteins / Q000737:chemistry*; D002614:Chelating Agents / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003300:Copper / Q000737:chemistry; D006801:Humans; D007098:Imino Acids / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D010455:Peptides / Q000097:blood* / Q000737:chemistry*; D011109:Polymethacrylic Acids / Q000737:chemistry*; D011145:Polyvinyls / Q000737:chemistry*; D040901:Proteomics / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D015415:Biomarkers; D001798:Blood Proteins; D002614:Chelating Agents; D007098:Imino Acids; D010455:Peptides; D011109:Polymethacrylic Acids; D011145:Polyvinyls; C516630:poly(glycidyl methacrylate-co-divinylbenzene); D003300:Copper; C008109:iminodiacetic acid