Quantification of fragments of human serum inter-alpha-trypsin inhibitor heavy chain 4 by a surface-enhanced laser desorption/ionization-based immunoassay.
BACKGROUND
Several proteolytically derived fragments from the proline-rich region (PRR) of human inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4) have been identified by surface-enhanced or matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS or MALDI-TOF-MS) as potential disease markers.


METHODS
Previously, we developed a SELDI-based immunoassay that can simultaneously distinguish and quantify multiple isoforms/variants of a protein/peptide of interest. In this study, we used this high-throughput approach to quantify and characterize the extensive fragmentation within the PRR of human serum ITIH4 and determined its association with different disease conditions. The ITIH4-related fragments were first immunocaptured by use of beads coupled with peptide-specific antibodies. The eluates were then studied by SELDI-TOF-MS. In addition, freshly collected and immediately processed serum and plasma samples were used to analyze the ex vivo stability of these ITIH4 fragments.


RESULTS
Human serum ITIH4 was shown to be extensively proteolytically processed within the PRR, and its fragmentation patterns were closely associated with different disease conditions. Fragmentation patterns were generally consistent with cleavages by endoprotease followed by exoprotease actions. Observed fragments changed little under different assay conditions or blood collection and processing procedures.


CONCLUSIONS
The fragmentation patterns within the PRR of human serum ITIH4 are associated with different disease conditions and may hold important diagnostic information. These fragmentation patterns could be useful as potential biomarkers for detection and classification of cancer.
Center for Biomarker Discovery, Department of Pathology, Johns Hopkins Medical Institutions, Baltimore, MD 21231, USA.
Jin
J
Song
Manisha
M
Patel
C Nicole
CN
Rosenzweig
Yee
Y
Chan-Li
Lori J
LJ
Sokoll
Eric T
ET
Fung
Nam-Ho
NH
Choi-Miura
Michael
M
Goggins
Daniel W
DW
Chan
Zhen
Z
Zhang
D000328:Adult; D000368:Aged; D000595:Amino Acid Sequence; D014408:Biomarkers, Tumor / Q000097:blood; D001798:Blood Proteins / Q000737:chemistry; D001943:Breast Neoplasms / Q000097:blood; D003110:Colonic Neoplasms / Q000097:blood; D003920:Diabetes Mellitus / Q000097:blood; D005260:Female; D006023:Glycoproteins / Q000097:blood* / Q000737:chemistry; D006801:Humans; D007118:Immunoassay; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D010051:Ovarian Neoplasms / Q000097:blood; D010190:Pancreatic Neoplasms / Q000097:blood; D011471:Prostatic Neoplasms / Q000097:blood; D053491:Proteinase Inhibitory Proteins, Secretory; D044967:Serum; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
D014408:Biomarkers, Tumor; D001798:Blood Proteins; D006023:Glycoproteins; C091236:ITIH4 protein, human; D053491:Proteinase Inhibitory Proteins, Secretory