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. 2025 Mar 25;10(13):13296-13302.
doi: 10.1021/acsomega.4c11051. eCollection 2025 Apr 8.

dia-PASEF Proteomics of Tumor and Stroma LMD Enriched from Archived HNSCC Samples

Aswini Panigrahi  1 , Allison L Hunt  2 , Diego Assis  3 , Matthew Willetts  3 , Bhaskar V Kallakury  4 , Bruce Davidson  5 , Jaeil Ahn  6 , Thomas P Conrads  2 , Radoslav Goldman  1   7
Affiliations

dia-PASEF Proteomics of Tumor and Stroma LMD Enriched from Archived HNSCC Samples

Aswini Panigrahi et al. ACS Omega. .

Abstract

We employed laser microdissection to selectively harvest histology-resolved tumors and stroma from formalin-fixed, paraffin-embedded head and neck squamous cell carcinoma (HNSCC) tissues. Peptide digests from the LMD-enriched HNSCC tissue were analyzed by quantitative mass-spectrometry-based proteomics using a data independent analysis approach. In paired samples, excellent proteome coverage was achieved, having quantified 6668 proteins with a median quantitative coefficient of variation under 10%. Significant differences in relevant functional pathways between the tumor and the stroma regions were observed. Extracellular matrix (ECM) was identified as a major component enriched in the stroma, including many cancer-associated fibroblast signature proteins in this compartment. We demonstrate the potential for comparative deep proteome analysis from a very low starting input in a scalable format. Correlating such results with clinical features or disease progression will likely enable the identification of novel targets for disease classification and interventions.

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Conflict of interest statement

The authors declare the following competing financial interest(s): AP, ALH, BVK, BD, JA, TPC, RG declare that they have no known competing financial interests. DA, MW are employees of Bruker Scientific.

Figures

Figure 1
Figure 1
Representative (A) H&E stained FFPE tissue sections on PEN membrane slides before (left) and after LMD harvest of tumor (middle) and stroma (right). Base peak chromatograms showing the overlay of triplicate runs of an enriched (B) tumor and (C) paired stroma sample. (D) Graphical representation of the number of proteins identified with 2 or more peptide matches in the enriched tumor and stroma samples. The median is indicated by a line. (E) Schematic representation of the CV value spread of quantitation for each sample; median (bold line) and upper and lower quartiles (thin line) are indicated.
Figure 2
Figure 2
Functional association of upregulated proteins. (A) Unsupervised hierarchical cluster analysis of stroma and tumor region proteins based on abundance. (B) Volcano plot of protein abundance. In volcano plot, the box dot plots of significantly changed proteins between the regions are indicated, and selected candidates are in red. Selected enriched functional pathways (String analysis) in stroma (C) and tumor (D) are shown with enrichment score and false discovery rate (FDR).
Figure 3
Figure 3
Protein–protein association map of stromal compartment upregulated (A) CAF markers and (B) heparin binding proteins.
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