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Heat ‘n Beat: A universal high-throughput end-to-end proteomics sample processing platform in under an hour

ABSTRACT: Clinical proteomics on solid tissue samples from human tumour biopsies present multiple challenges. These include limited sample availability, reducing endogenous protease activity and challenges with efficiently solubilising and digesting proteins for high throughput mass spectrometry while achieving comprehensive proteome coverage. Protocols for cancer biopsy proteomics involves three sample preparation steps: tissue washing, lysis and digestion, followed by peptide clean-up by SPE. Our team have reported improvements in all three steps that greatly reduce processing time, enabling greater automation and higher sample throughput. Our aim was to develop a universal protocol for tissue sample preparation that is quick and simple while improving protein solubilization, proteome coverage and reproducibility. Sample preparation with small (<2 mg) biopsies pose additional limitations including the risk of sample loss and introducing variability with multiple reagent additions steps. We now report a one-pot method called heat and beat (HnB). By identifying the limitations and challenges of the existing protocols, we were able to develop a new protocol with fewer steps by achieving lysis, reduction, alkylation and digestion in essentially a single step which was preceded by a 7 min sample heat treatment step. This was achieved by several changes to the existing protocol including the combined use of a beadbeater for tissue homogenisation followed by pressure cycling technology (PCT) using a Barocycler to further lyse and digest the sample. The procedure requires 38 minutes and was tested on 6 tissue types, across three tissue embedding techniques, fresh frozen, OCT embedded (FF-OCT), and FFPE. Our results show that HnB is an efficient new one-pot procedure. The protocol significantly reduced endogenous protease activity, improves peptide yield by up to 3-fold and delivers digestion efficiency of 85-90% from each sample source. We obtained an increase in the number of proteins identified of up to two-fold while reducing variability and processing time.

INSTRUMENT(S): TripleTOF 6600

ORGANISM(S): Rattus Norvegicus (rat) Homo Sapiens (human)

TISSUE(S): Testis, Brain, Liver, Lung, Cell Culture, Muscle, Kidney

SUBMITTER: Dylan Xavier

LAB HEAD: Phillip J. Robinson

PROVIDER: PXD045405 | Pride | 2025-03-26

REPOSITORIES: Pride

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			Action	DRS
	171130_FFPE_1_I_M01_1.group	Other
	171130_FFPE_1_I_M01_1.wiff	Wiff
	171130_FFPE_1_I_M01_1.wiff.scan	Wiff
	171130_FFPE_1_I_M01_1__FDR.xlsx	Xlsx
	171130_FFPE_2_I_M01_1.group	Other

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Project description:Tissue samples in biobanks preserved by formalin-fixation and paraffin-embedding (FFPE) and/or optimal cutting temperature (OCT)-embedded and subsequently freezing are highly valuable in experimental studies where the results can be related to clinical parameters. Today, mass spectrometry (MS)-based proteomics is the method of choice for unbiased and relative comparison of the abundances of thousands of proteins present in biological samples. MS analysis of FFPE- and OCT-preserved tissue has been limited, but it is now approachable using newly developed protocols. However, it has not been clarified how the results from different preservation methods agrees and differ. In this study, we use a unique sample set of urinary bladder cancer tissues of two different tumor stages (Ta/T1 – non-muscle invasive and T2/T3 muscle invasive); upon sampling the tumors were divided and preserved by the two methods. Thereafter a protocol for parallel processing of the samples were applied after which the samples were analyzed with label free quantitative MS analysis. Over 700 and 1200 proteins were quantified in FFPE and OCT samples, respectively. Principal component analysis showed that the largest differences between samples are based on the preservation method, but this analysis could also classify the tumors into different stages in each preservation. The overlap of quantifiable proteins between the preservation methods were 84% when all proteins and full data set were taken into consideration, but only 41% on average when proteins from a specific tumor were analyzed. Proteins involved in mitochondrial function were overrepresented among proteins present in OCT data but missing in the FFPE data, indicating that these proteins are not well preserved by FFPE. Moreover, univariate analysis showed that HMGCS2 protein is uniquely quantified in Ta/T1 tumors in both FFPE and OCT data, and that LGALS1, ANXA5 and plastin proteins are upregulated in T2/T3 tumors compared to Ta/T1 tumors in both FFPE and OCT data, confirming the consistency between the data sets and suggesting these proteins as biomarkers. Finally, LGALS1 data were verified by immunohistochemistry staining of an independent data set.

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Heat ‘n Beat: A universal high-throughput end-to-end proteomics sample processing platform in under an hour

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