A Comparison of Different Sample Processing Protocols for MALDI Imaging Mass Spectrometry Analysis of Formalin-Fixed Multiple Myeloma Cells

SIMPLE SUMMARY: Appropriate sample preparation is critical for the analysis of cell cultures with mass spectrometry. It is important not only to maintain clear and consistent morphological features, but also the local chemical composition of the sample. In this proof-of-concept study, we evaluated two different sample preparation procedures for proteomic analysis using imaging mass spectrometry (IMS) of formalin-fixed multiple myeloma (MM)-cultured cells. Cytospin preparation resulted in more peaks with a signal-to-noise ratio > 3 as compared to formalin fixation and paraffin embedding. Overall, IMS technology holds the potential to stratify different cell lines to address the identification of differentially expressed proteins. We propose this approach as an additional feasible method for proteomic investigation of MM cell lines, and potentially applicable to other tumor types. ABSTRACT: Sample processing of formalin-fixed specimens constitutes a major challenge in molecular profiling efforts. Pre-analytical factors such as fixative temperature, dehydration, and embedding media affect downstream analysis, generating data dependent on technical processing rather than disease state. In this study, we investigated two different sample processing methods, including the use of the cytospin sample preparation and automated sample processing apparatuses for proteomic analysis of multiple myeloma (MM) cell lines using imaging mass spectrometry (IMS). In addition, two sample-embedding instruments using different reagents and processing times were considered. Three MM cell lines fixed in 4% paraformaldehyde were either directly centrifuged onto glass slides using cytospin preparation techniques or processed to create paraffin-embedded specimens with an automatic tissue processor, and further cut onto glass slides for IMS analysis. The number of peaks obtained from paraffin-embedded samples was comparable between the two different sample processing instruments. Interestingly, spectra profiles showed enhanced ion yield in cytospin compared to paraffin-embedded samples along with high reproducibility compared to the sample replicate.