Identification of blood biomarkers in glioblastoma by SWATH mass spectrometry and quantitative targeted absolute proteomics

<div><p>Molecular biomarkers in blood are needed to aid the early diagnosis and clinical assessment of glioblastoma (GBM). Here, in order to identify biomarker candidates in plasma of GBM patients, we performed quantitative comparisons of the plasma proteomes of GBM patients (n = 14) and healthy controls (n = 15) using SWATH mass spectrometry analysis. The results were validated by means of quantitative targeted absolute proteomics analysis. As a result, we identified eight biomarker candidates for GBM (leucine-rich alpha-2-glycoprotein (LRG1), complement component C9 (C9), C-reactive protein (CRP), alpha-1-antichymotrypsin (SERPINA3), apolipoprotein B-100 (APOB), gelsolin (GSN), Ig alpha-1 chain C region (IGHA1), and apolipoprotein A-IV (APOA4)). Among them, LRG1, C9, CRP, GSN, IGHA1, and APOA4 gave values of the area under the receiver operating characteristics curve of greater than 0.80. To investigate the relationships between the biomarker candidates and GBM biology, we examined correlations between plasma concentrations of biomarker candidates and clinical presentation (tumor size, progression-free survival time, or overall survival time) in GBM patients. The plasma concentrations of LRG1, CRP, and C9 showed significant positive correlations with tumor size (R² = 0.534, 0.495, and 0.452, respectively).</p></div

Box plot showing the plasma levels of each differentially expressed protein in GBM plasma (n = 14) compared with healthy plasma (n = 15).

<p>Each dot represents the protein level of an individual sample. In box plots, the band inside the box represents the median. The bottom and top of the box represent the first and third quartiles. The whiskers reflect the minimum and maximum values that fall within 1.5 times the interquartile range. Any data not included between the whiskers is an outlier. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Cont, Healthy controls; GBM, glioblastoma.</p

Summary of the differentially expressed proteins in plasma validated by QTAP analysis.

<p>Summary of the differentially expressed proteins in plasma validated by QTAP analysis.</p

Characteristics of subjects.

<p>Characteristics of subjects.</p

Identification of blood biomarkers in glioblastoma by SWATH mass spectrometry and quantitative targeted absolute proteomics - Fig 3

<p><b>Kaplan–Meier curve of progression-free survival time (PFS) (A) and overall survival time (OS) (B) in patients with glioblastoma (GBM) showed prognostic significance of gelsolin (GSN).</b> GBM patients were classified into two categories on the basis of GSN level: low (0–472 fmol/μL plasma) and high (> 472 fmol/μL plasma). Mean GSN plasma level in GBM patients was selected as the cut-off point. (A) PFS interval was determined as the interval between the date of initial operation and the date of patient’s recurrence or determined endpoint (for those no recurrent on August 1, 2015). (B) OS interval was determined as the interval between the date of the initial operation and date of patient’s death or determined end point (for those alive on August 1, 2015).</p

Total number of proteins and peptides in spectral library.

<p>Total number of proteins and peptides in spectral library.</p

Flow diagram of GBM biomarker discovery.

<p>Flow diagram of GBM biomarker discovery.</p

Summary of the differentially expressed proteins identified in plasma analyzed by SWATH-MS analysis.

<p>Summary of the differentially expressed proteins identified in plasma analyzed by SWATH-MS analysis.</p