Isobaric Labeling Strategy Utilizing 4-Plex N, N-Dimethyl Leucine (DiLeu) Tags Reveals Proteomic Changes Induced by Chemotherapy in Cerebrospinal Fluid of Children with B-Cell Acute Lymphoblastic Leukemia

Abstract

The use of mass spectrometry for protein identification and quantification in cerebrospinal fluid (CSF) is at the forefront of research efforts to identify and explore biomarkers for the early diagnosis and prognosis of neurologic disorders. Here we implemented a 4-plex N,N-dimethyl leucine (DiLeu) isobaric labeling strategy in a longitudinal study aiming to investigate protein dynamics in children with B-cell acute lymphoblastic leukemia (B-cell ALL) undergoing chemotherapy. The temporal profile of CSF proteome during chemotherapy treatment at weeks 5, 10-14, and 24-28 highlighted many differentially expressed proteins, such as neural cell adhesion molecule, neuronal growth regulator 1, and secretogranin-3, all of which play important roles in neurodegenerative diseases. A total of 63 proteins were significantly altered across all of the time points investigated. The most over-represented biological processes from gene ontology analysis included platelet degranulation, complement activation, cell adhesion, fibrinolysis, neuron projection, regeneration, and regulation of neuron death. We expect that results from this and future studies will provide a means to monitor neurotoxicity and develop strategies to prevent central nervous system injury in response to chemotherapy in children.

Keywords: B-cell acute lymphoblastic leukemia; central nervous system; cerebrospinal fluid; chemotherapy; dimethyl leucine (DiLeu) isobaric labeling; protein dynamics.

Figure 1.

Experimental workflow for multiplexed CSF protein quantification. CSF samples were collected from 9 patients before chemotherapy at week 1 and at weeks 5, 10–14, 24–28 during chemotherapy. For each patient, samples were digested with trypsin/Lys-C, labeled with 4-plex DiLeu tags respectively, and combined for sample clean-up with SCX SpinTips. Samples were analyzed using a nanoLC system coupled to a Q-Exactive HF Orbitrap mass spectrometer. Data was searched through Proteome Discoverer and further processed by Perseus and R.

Figure 2.

Volcano plots showed CSF protein level changes between (A) week 5 vs. week 1; (B) week 10–14 vs. week 1; and (C) week 24–28 vs. week 1. Log₂ protein fold changes are plotted against the negative log₁₀ adjusted p-values (FDR) from nine biological replicates. Points above the non-axial horizontal line represent significantly altered proteins (lower line, adjusted p-value < 0.1; upper line, adjusted p-value < 0.05, student’s t-test). Green dots represent proteins with decreased levels and red dots represent proteins with increased levels.

Figure 3.

(A) Hierarchical clustering of the 63 proteins that exhibited statistically significant expression (ANOVA test, adjusted p-value < 0.1) in the CSF visualized in a heatmap. (B) Expression profile of the two hierarchical clusters, which were statistically different relative to one another (color based on distance from center in Perseus).

Figure 4.

Proteins to biological pathway linkages for proteins in (A) cluster 1 and (B) cluster 2 based on hierarchical clustering analysis. The significantly altered proteins (ANOVA, adjusted p-value < 0.1) have been linked with pathways as color-coded ribbons. Green-to-red rectangles next to the altered proteins indicate the magnitude of the log₂ fold change (FC), where the inner layer represents FC of week 1 in comparison with week 5, the middle layer in comparison with week 10–14 and the outer layer in comparison with week 24–28.

Figure 5.

Box plot of the distribution of (A) neural cell adhesion molecule 2; (B) neuronal cell adhesion molecule; (C) neuronal growth regulator 1 and (D) secretogranin-3 in CSF at different stages of chemotherapy. Whiskers extend to data points that are less than 1.5* inter-quartile range away from 1^st and 3^rd quartile, respectively. The horizontal line shows the median. (*, adjusted p-value < 0.1; **, adjusted p-value < 0.05, Student’s t-test)