Analysis of the low molecular weight serum peptidome using ultrafiltration and a hybrid ion trap-Fourier transform mass spectrometer.
Advances in proteomics are continuing to expand the ability to analyze the serum proteome. In recent years, it has been realized that in addition to the circulating proteins, human serum also contains a large number of peptides. Many of these peptides are believed to be fragments of larger proteins that have been at least partially degraded by various enzymes such as metalloproteases. Identifying these peptides from a small amount of serum/plasma is difficult due to the complexity of the sample, the low levels of these peptides, and the difficulties in getting a protein identification from a single peptide. In this study, we modified previously published protocols for using centrifugal ultrafiltration, and unlike past studies did not digest the filtrate with trypsin with the intent of identifying endogenous peptides with this method. The filtrate fraction was concentrated and analyzed by a reversed phase-high performance liquid chromatography system connected to a nanospray ionization hybrid ion trap-Fourier transform mass spectrometer (LTQ-FTMS). The mass accuracy of this instrument allows confidence for identifying the protein precursors by a single peptide. The utility of this approach was demonstrated by the identification of over 300 unique peptides with 2 ppm or better mass accuracy per serum sample. With confident identifications, the origin and function of native serum peptides can be more seriously explored. Interestingly, over 34 peptide ladders were observed from over 17 serum proteins. This indicates that a cascade of proteolytic processes affects the serum peptidome. To examine whether this result was an artifact of serum, matched plasma and serum samples were analyzed with similar peptide ladders found in each.
Zheng
Xiaoyang
X
Barnett Institute and Department of Chemistry, Northeastern University, 341 Mugar Hall, 360 Huntington Avenue, Boston, MA 02115, USA.
Baker
Haven
H
Hancock
William S
WS
D000595:Amino Acid Sequence; D001798:Blood Proteins / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D030562:Databases, Protein; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis* / Q000097:blood / Q000737:chemistry; D040901:Proteomics / Q000379:methods; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods*; D014462:Ultrafiltration / Q000379:methods
D001798:Blood Proteins; D010446:Peptide Fragments