Abstract
BackgroundProteomic studies are typically conducted using flash-frozen (FF) samples utilizing tandem mass spectrometry (MS). However, FF specimens are comprised of multiple cell types, making it difficult to ascertain the proteomic profiles of specific cells. Conversely, OCT-embedded (Optimal Cutting Temperature compound) specimens can undergo laser microdissection (LMD) to capture and study specific cell types separately from the cell mixture. In the current study, we compared proteomic data obtained from FF and OCT samples to determine if samples that are stored and processed differently produce comparable results.MethodsProteins were extracted from FF and OCT-embedded invasive breast tumors from 5 female patients. FF specimens were lysed via homogenization (FF/HOM) while OCT-embedded specimens underwent LMD to collect only tumor cells (OCT/LMD-T) or both tumor and stromal cells (OCT/LMD-TS) followed by incubation at 37 °C. Proteins were extracted using the illustra triplePrep kit and then trypsin-digested, TMT-labeled, and processed by two-dimensional liquid chromatography-tandem mass spectrometry (2D LC–MS/MS). Proteins were identified and quantified with Proteome Discoverer v1.4 and comparative analyses performed to identify proteins that were significantly differentially expressed amongst the different processing methods.ResultsAmong the 4,950 proteins consistently quantified across all samples, 216 and 171 proteins were significantly differentially expressed (adjusted p-value < 0.05; |log2 FC|> 1) between FF/HOM vs. OCT/LMD-T and FF/HOM vs. OCT/LMD-TS, respectively, with most proteins being more highly abundant in the FF/HOM samples. PCA and unsupervised hierarchical clustering analysis with these 216 and 171 proteins were able to distinguish FF/HOM from OCT/LMD-T and OCT/LMD-TS samples, respectively. Similar analyses using significantly differentially enriched GO terms also discriminated FF/HOM from OCT/LMD samples. No significantly differentially expressed proteins were detected between the OCT/LMD-T and OCT/LMD-TS samples but trended differences were detected.ConclusionsThe proteomic profiles of the OCT/LMD-TS samples were more similar to those from OCT/LMD-T samples than FF/HOM samples, suggesting a strong influence from the sample processing methods. These results indicate that in LC–MS/MS proteomic studies, FF/HOM samples exhibit different protein expression profiles from OCT/LMD samples and thus, results from these two different methods cannot be directly compared.
Highlights
Proteomic studies are typically conducted using flash-frozen (FF) samples utilizing tandem mass spectrometry (MS)
The proteomic profiles of the Optimal cutting temperature compound (OCT)/laser microdissection (LMD)-TS samples were more similar to those from OCT/Laser microdissection of tumor only (LMD-T) samples than FF specimens were lysed via homogenization (FF/HOM) samples, suggesting a strong influence from the sample processing methods. These results indicate that in LC–MS/MS proteomic studies, FF/HOM samples exhibit different protein expression profiles from OCT/ LMD samples and results from these two different methods cannot be directly compared
Number of detected proteins across samples To evaluate the impact of sample storage and preparation methods for breast tumor tissues on quantitative proteomic analysis, we compared proteins quantified from five breast tumor specimens processed by two methods in three different ways: FF/HOM, OCT/LMD-T and OCT/LMD-TS (Fig. 1)
Summary
Proteomic studies are typically conducted using flash-frozen (FF) samples utilizing tandem mass spectrometry (MS). OCT-embedded (Optimal Cutting Temperature compound) specimens can undergo laser microdissection (LMD) to capture and study specific cell types separately from the cell mixture. Clinical tissue specimens are a valuable resource for cancer research and can be preserved using multiple types of storage media. Tissue specimens may be preserved by embedding them in OCT (Optimal Cutting Temperature) compound. Both FF and OCT-embedded specimens yield highquality nucleic acids and proteins for downstream molecular analyses. FF has been the preferred storage method for protein extraction and processing as OCT can interfere with the performance of liquid chromatography-tandem mass spectrometry (LC–MS/MS) [1]. Several methods to remove OCT have been published that have resulted in an improved number of protein identifications by MS [1,2,3,4,5]
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