Evaluation of serum phosphopeptides as potential cancer biomarkers by mass spectrometric absolute quantification.
Mass spectrometric quantification of phosphopeptides is a challenge due to the ion suppression effect of highly abundant non-phosphorylated peptides in complex samples such as serum. Several strategies for relative quantification of serum phosphopeptides based on MS have been developed, but the power of relative quantities was limited when making direct comparisons between two groups of samples or acting as a clinical examination index. Herein, we describe an MS absolute quantification strategy combined with Titania Coated Magnetic Hollow Mesoporous Silica Microspheres (TiO2/MHMSM) enrichment and stable isotopic acetyl labeling for phosphopeptides in human serum. Four endogenous serum phosphopeptides generated by degradation of fibrinogen were identified by LC-ESI-MS/MS following TiO2/MHMSM enrichment. The ESI-MS signal intensity ratios of the four phosphopeptide standards labeled with N-acetoxy-H3-succinimide (H3-NAS) and N-acetoxy-D3-succinimide (D3-NAS), following TiO2/MHMSM capture are linearly correlated with the molar ratios of the "light" to "heavy" phosphopeptides over the range of 0.1-4 with an r(2) of up to 0.998 and a slope of close to 1. The recovery of the four phosphopeptides spiked at low, medium and high levels in human sera were 98.4-111.9% with RSDs ranging 2.0-10.1%. The absolute quantification of the phosphopeptides in serum samples of 20 healthy persons and 20 gastric cancer patients by the developed method demonstrated that 3 out of the 4 phosphopeptides showed remarkable variation in serum level between healthy and cancer groups, and the phosphopeptide DpSGEGDFLAEGGGVR is significantly down-regulated in the serum of patients, being a potential biomarker for gastric cancer diagnosis.
Beijing National Laboratory for Molecular Sciences; CAS Key Laboratory of Analytical Chemistry for Living Biosystems; Beijing Center for Mass Spectrometry; Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, PR China.
Guijin
G
Zhai
Cancer Research Center, Tumor Hospital Affiliated to Nantong University, Nantong 226361, Jiangsu, PR China.
Xiaoyan
X
Wu
Beijing National Laboratory for Molecular Sciences; CAS Key Laboratory of Analytical Chemistry for Living Biosystems; Beijing Center for Mass Spectrometry; Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, PR China.
Qun
Q
Luo
Beijing National Laboratory for Molecular Sciences; CAS Key Laboratory of Analytical Chemistry for Living Biosystems; Beijing Center for Mass Spectrometry; Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, PR China.
Kui
K
Wu
Beijing National Laboratory for Molecular Sciences; CAS Key Laboratory of Analytical Chemistry for Living Biosystems; Beijing Center for Mass Spectrometry; Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, PR China.
Yao
Y
Zhao
Beijing National Laboratory for Molecular Sciences; CAS Key Laboratory of Analytical Chemistry for Living Biosystems; Beijing Center for Mass Spectrometry; Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, PR China.
Jianan
J
Liu
Beijing National Laboratory for Molecular Sciences; CAS Key Laboratory of Analytical Chemistry for Living Biosystems; Beijing Center for Mass Spectrometry; Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, PR China.
Shaoxiang
S
Xiong
College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, PR China.
Yu-Qi
YQ
Feng
Cancer Research Center, Tumor Hospital Affiliated to Nantong University, Nantong 226361, Jiangsu, PR China. Electronic address: liping.yang@ntu.edu.cn.
Liping
L
Yang
Beijing National Laboratory for Molecular Sciences; CAS Key Laboratory of Analytical Chemistry for Living Biosystems; Beijing Center for Mass Spectrometry; Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, PR China. Electronic address: fuyi.wang@iccas.ac.cn.
Fuyi
F
Wang
D014408:Biomarkers, Tumor / Q000097:blood*; D015536:Down-Regulation; D006801:Humans; D008863:Microspheres; D009369:Neoplasms / Q000097:blood*; D010455:Peptides / Q000737:chemistry; D010748:Phosphopeptides / Q000097:blood* / Q000737:chemistry*; D012372:ROC Curve; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013274:Stomach Neoplasms / Q000097:blood*; D014025:Titanium / Q000737:chemistry
D014408:Biomarkers, Tumor; D010455:Peptides; D010748:Phosphopeptides; C009495:titanium dioxide; D014025:Titanium
Absolute quantification; Cancer biomarker; Enrichment; Mass spectrometry; Serum phosphopeptides