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Rapid mass spectrometric conversion of tissue biopsy samples into permanent quantitative digital proteome maps.

Guo T, Kouvonen P, Koh CC, Gillet LC, Wolski WE, Röst HL, Rosenberger G, Collins BC, Blum LC, Gillessen S, Joerger M, Jochum W, Aebersold R - Nat. Med. (2015)

Bottom Line: The method combines pressure cycling technology (PCT) and sequential window acquisition of all theoretical fragment ion spectra (SWATH)-MS.The resulting proteome maps can be analyzed, re-analyzed, compared and mined in silico to detect and quantify specific proteins across multiple samples.From these proteome maps we detected and quantified more than 2,000 proteins with a high degree of reproducibility across all samples.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Institute of Molecular Systems Biology, Eidgenössische Technische Hochschule (ETH) Zurich, Zurich, Switzerland.

ABSTRACT
Clinical specimens are each inherently unique, limited and nonrenewable. Small samples such as tissue biopsies are often completely consumed after a limited number of analyses. Here we present a method that enables fast and reproducible conversion of a small amount of tissue (approximating the quantity obtained by a biopsy) into a single, permanent digital file representing the mass spectrometry (MS)-measurable proteome of the sample. The method combines pressure cycling technology (PCT) and sequential window acquisition of all theoretical fragment ion spectra (SWATH)-MS. The resulting proteome maps can be analyzed, re-analyzed, compared and mined in silico to detect and quantify specific proteins across multiple samples. We used this method to process and convert 18 biopsy samples from nine patients with renal cell carcinoma into SWATH-MS fragment ion maps. From these proteome maps we detected and quantified more than 2,000 proteins with a high degree of reproducibility across all samples. The measured proteins clearly distinguished tumorous kidney tissues from healthy tissues and differentiated distinct histomorphological kidney cancer subtypes.

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Related in: MedlinePlus

Yield and efficiency of PCT-assisted tissue lysis and protein digestion(a) Ten aliquots of U2OS cells, four aliquots of mouse liver tissue, and twelve aliquots of human kidney tissue were processed with the PCT method. The final peptide yield was normalized to micrograms of peptides per million cells or milligram of tissue, respectively. Yields are shown in box-and-whiskers plots. The boundaries of the box denote 25% and 75% quantiles, whereas the horizontal line in the middle of the box indicates the median. Whiskers indicate the quantile boundaries for values beyond 1.5 times the interquartile range. Small circle beyond whiskers indicates outlier. (b) Mouse liver tissue was digested with duplicates using the PCT and a conventional (CONV) method. The produced peptides were analyzed by DDA mass spectrometry. Ratios of PCT and CONV in terms of a few parameters are displayed.
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Figure 2: Yield and efficiency of PCT-assisted tissue lysis and protein digestion(a) Ten aliquots of U2OS cells, four aliquots of mouse liver tissue, and twelve aliquots of human kidney tissue were processed with the PCT method. The final peptide yield was normalized to micrograms of peptides per million cells or milligram of tissue, respectively. Yields are shown in box-and-whiskers plots. The boundaries of the box denote 25% and 75% quantiles, whereas the horizontal line in the middle of the box indicates the median. Whiskers indicate the quantile boundaries for values beyond 1.5 times the interquartile range. Small circle beyond whiskers indicates outlier. (b) Mouse liver tissue was digested with duplicates using the PCT and a conventional (CONV) method. The produced peptides were analyzed by DDA mass spectrometry. Ratios of PCT and CONV in terms of a few parameters are displayed.

Mentions: To assess the yield and reproducibility of the optimized PCT method, we determined the total peptide amount generated from a human cell line and two types of tissues. In every case, the total amount of processed, MS injection-ready peptide was determined based on its absorbance at 280 nm using NanoDrop. From the human osteosarcoma cell line U2OS, approximately 116 μg of total peptide mass was generated per million cells (Fig. 2a). From aliquots of 1-3 milligram samples of wet mouse liver and human kidney tissue, the method produced about 50 μg of total peptide mass per milligram of tissue with a high degree of reproducibility for technical replicates (coefficient of variation, CV < 8%). The CV of peptide yield increased to 41% for 12 human kidney tissues from different patients, probably due to biological variation.


Rapid mass spectrometric conversion of tissue biopsy samples into permanent quantitative digital proteome maps.

Guo T, Kouvonen P, Koh CC, Gillet LC, Wolski WE, Röst HL, Rosenberger G, Collins BC, Blum LC, Gillessen S, Joerger M, Jochum W, Aebersold R - Nat. Med. (2015)

Yield and efficiency of PCT-assisted tissue lysis and protein digestion(a) Ten aliquots of U2OS cells, four aliquots of mouse liver tissue, and twelve aliquots of human kidney tissue were processed with the PCT method. The final peptide yield was normalized to micrograms of peptides per million cells or milligram of tissue, respectively. Yields are shown in box-and-whiskers plots. The boundaries of the box denote 25% and 75% quantiles, whereas the horizontal line in the middle of the box indicates the median. Whiskers indicate the quantile boundaries for values beyond 1.5 times the interquartile range. Small circle beyond whiskers indicates outlier. (b) Mouse liver tissue was digested with duplicates using the PCT and a conventional (CONV) method. The produced peptides were analyzed by DDA mass spectrometry. Ratios of PCT and CONV in terms of a few parameters are displayed.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4390165&req=5

Figure 2: Yield and efficiency of PCT-assisted tissue lysis and protein digestion(a) Ten aliquots of U2OS cells, four aliquots of mouse liver tissue, and twelve aliquots of human kidney tissue were processed with the PCT method. The final peptide yield was normalized to micrograms of peptides per million cells or milligram of tissue, respectively. Yields are shown in box-and-whiskers plots. The boundaries of the box denote 25% and 75% quantiles, whereas the horizontal line in the middle of the box indicates the median. Whiskers indicate the quantile boundaries for values beyond 1.5 times the interquartile range. Small circle beyond whiskers indicates outlier. (b) Mouse liver tissue was digested with duplicates using the PCT and a conventional (CONV) method. The produced peptides were analyzed by DDA mass spectrometry. Ratios of PCT and CONV in terms of a few parameters are displayed.
Mentions: To assess the yield and reproducibility of the optimized PCT method, we determined the total peptide amount generated from a human cell line and two types of tissues. In every case, the total amount of processed, MS injection-ready peptide was determined based on its absorbance at 280 nm using NanoDrop. From the human osteosarcoma cell line U2OS, approximately 116 μg of total peptide mass was generated per million cells (Fig. 2a). From aliquots of 1-3 milligram samples of wet mouse liver and human kidney tissue, the method produced about 50 μg of total peptide mass per milligram of tissue with a high degree of reproducibility for technical replicates (coefficient of variation, CV < 8%). The CV of peptide yield increased to 41% for 12 human kidney tissues from different patients, probably due to biological variation.

Bottom Line: The method combines pressure cycling technology (PCT) and sequential window acquisition of all theoretical fragment ion spectra (SWATH)-MS.The resulting proteome maps can be analyzed, re-analyzed, compared and mined in silico to detect and quantify specific proteins across multiple samples.From these proteome maps we detected and quantified more than 2,000 proteins with a high degree of reproducibility across all samples.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Institute of Molecular Systems Biology, Eidgenössische Technische Hochschule (ETH) Zurich, Zurich, Switzerland.

ABSTRACT
Clinical specimens are each inherently unique, limited and nonrenewable. Small samples such as tissue biopsies are often completely consumed after a limited number of analyses. Here we present a method that enables fast and reproducible conversion of a small amount of tissue (approximating the quantity obtained by a biopsy) into a single, permanent digital file representing the mass spectrometry (MS)-measurable proteome of the sample. The method combines pressure cycling technology (PCT) and sequential window acquisition of all theoretical fragment ion spectra (SWATH)-MS. The resulting proteome maps can be analyzed, re-analyzed, compared and mined in silico to detect and quantify specific proteins across multiple samples. We used this method to process and convert 18 biopsy samples from nine patients with renal cell carcinoma into SWATH-MS fragment ion maps. From these proteome maps we detected and quantified more than 2,000 proteins with a high degree of reproducibility across all samples. The measured proteins clearly distinguished tumorous kidney tissues from healthy tissues and differentiated distinct histomorphological kidney cancer subtypes.

Show MeSH
Related in: MedlinePlus