Endogenous peptides from biophysical and biochemical fractionation of serum analyzed by matrix-assisted laser desorption/ionization and electrospray ionization hybrid quadrupole time-of-flight

• Published in: Analytical biochemistry Vol. 370; no. 2; pp. 228 - 245
• Main Authors: Tucholska, Monika, Scozzaro, Salvatore, Williams, Declan, Ackloo, Suzanne, Lock, Chris, Siu, K.W. Michael, Evans, Kenneth R., Marshall, John G.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15.11.2007
• Subjects: Amino Acid Sequence; Amyloid and coagulation factor; Blood Proteins - chemistry; Chromatography, Gel; Electrophoresis, Polyacrylamide Gel; Extracellular matrix; Humans; LC–ESI–Qq–TOF; MALDI; Mass Spectrometry - methods; Molecular Sequence Data; Peptides - blood; Peptides - isolation & purification; Peptides in serum; Spectrometry, Mass, Electrospray Ionization - methods; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods; Thymosin; LC–ESI–Qq–TOF; Thymosin; Amyloid and coagulation factor; Extracellular matrix; Peptides in serum; MALDI
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2007.07.029

Blood peptides can be concentrated, extracted, and analyzed with strong signal-to-noise ratios by precipitation in organic solvents followed by extraction in water. Matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) hybrid quadrupole time-of-flight (Qq–TOF) were used to analyze the precipitated and extracted endogenous peptides from fetal calf serum. C18 solid-phase extraction with or without prior precipitation in ammonium sulfate, size exclusion chromatography, dealbuminization, dye affinity chromatography, ultrafiltration, and differential precipitation in organic solvents were compared. Hundreds of different ions could be observed by MALDI in the various fractions. It appeared that some peptides were freely dissolved and that not all peptides in blood were obliged to remain bound to albumin or other high-molecular-mass proteins. Mass spectra with high signal-to-noise ratios were obtained from polypeptides precipitated with organic solvents followed by extraction of the peptides from the pellet with water. The peptides extracted from organic precipitates were analyzed by nano liquid chromatography (LC)–ESI–Qq–TOF. In addition to many commonly abundant serum proteins, apparent low-abundance peptides associated with cancer biology from proteins such as insulin-like growth factor II, thymosin β4 and β9, plasminogen, coagulation factors, and extracellular matrix protein 1 were observed.