Page 26 - Read Online

Ossoliński et al. J Cancer Metastasis Treat 2019;5:1 I http://dx.doi.org/10.20517/2394-4722.2018.63 Page 5 of 12

was 1000 Hz; and the deflection value was set to m/z 80 Da. The first accelerating voltage was held at 19 kV and
the second ion-source voltage was held at 16.7 kV. The reflector voltages used were 21 kV (first) and 9.55 kV
(second). Measurement range was m/z 80-2000.

Samples were placed on AuNPET plate semi-automatically with the aid of precise positioning of 3D
translation stage which allowed precise orientation of commonly used pipette (Eppendorf Research plus).
Application of described stage allowed much higher spot density and repeatability which would be rather
impossible in manual manner. Semi-automated solution applied allowed measurements of over hundreds of
samples on one custom-made target plates of 3.5 × 2.5 cm size.

For each sample spot, four measurement locations (pixels) were measured and resulting spectra averaged
after preliminary re-calibration. Obtained quadruplicates were additionally recalibrated and normalized, the
latter was based on three most abundant gold ion peaks.

Extracts were measured in MSI mode with 20,000 laser shots per individual pixel and four pixels per sample
with 300 µm-lateral resolution. Bruker’s FlexImaging 4.0 software was used for setting of MSI experiments.
+
+
Mass calibration was performed using internal standards (gold ions and clusters from Au to Au ). Imaging
9
data were converted to Analyze 7.5 and then to mzXML format and processed in mMass software. Obtained
spectra were processed by application of baseline correction and recalibration. Spectra from four pixels of
each spot were averaged and then again recalibrated. Intensities of averaged spectra of spots were normalized
+
+
+
based on arithmetical mean of intensities of Au , Au and Au ions with the use of software developed
3
2
by our group. Resulting ion lists were then assigned with the use of monoisotopic masses available for
metabolites enclosed in Human Metabolome Database (HMDB) and Pathos database within 10 ppm mass
accuracy windows.
Study design
Serum and urine were collected from all the patients and categorized as follows: (1) no PCa (healthy
volunteers, n = 10 patients); (2) presumptive benign disease (negative biopsy, n = 5 patients); (3) PCa
(positive biopsy, n = 5 patients). IF collected from each prostate biopsy core was categorized as follows: (1)
presumptive benign disease (negative biopsy, n = 5 patients); (2) PCa (positive biopsy, n = 5 patients). Each
sample was analyzed using standardized MS protocol. Serum and urine specimens collected from patients
with biopsy confirmed PCa were compared with those from healthy volunteers (no biopsy in this group).
Serum and urine from patients with negative biopsy (no PCa) were excluded from this study due to lack
of certainty that there is no PCa in non-biopsied regions of prostate. IF from positive biopsy cores were
compared with those from negative cores. Patient characteristics are shown in Table 1.

RESULTS
All statistically significant metabolites (P < 0.05) are shown in Table 2. Statistical analysis was performed
using SPSS and R software package. Four, twenty-two and ten metabolites from urine, serum and IF
respectively showed statistical significant (P < 0.05) differential abundance between cancer and control
group via Mann-Whitney U test. In addition, metabolites with m/z values of 897.7 (urine) and 307.0 (IF)
were positively correlated with prostate volume (Spearman’s rho = 0.564, P = 0.028 and 0.648, P = 0.043
respectively). Differences between cancer/control intensities have been presented in the form of box- [Figure 1]
and volcano-plot [Figure 2] graphs. Peaks obtained from MS analyses were identified and analyzed in the
context of metabolic pathways in which they appear within Pathos and HMDB.

DISCUSSION
In this study, a new methodology for rapid metabolomic analysis of human physiological liquids is proposed.
MS measurements in imaging mode based on LDI apparatus, with the use of AuNPET target plate, simplify