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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.The measured proteins clearly distinguished tumorous kidney tissues from healthy tissues and differentiated distinct histomorphological kidney cancer subtypes.

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

Reproducibility of PCT-SWATH(a) Four aliquots of a human kidney tissue sample were separately digested using PCT, and each analyzed by SWATH-MS in duplicates. The distribution of CV values of protein quantification for each digestion batch, injection batch, and total variation is shown as violin plots where the kernel density curves shape the violin, the white node in the center denotes median value, and the black box inside the violin shows the same interquantile range as the box-and-whiskers plot. (b) Bar plot of average median CV of injection, digestion and the entire workflow (Total).
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Figure 3: Reproducibility of PCT-SWATH(a) Four aliquots of a human kidney tissue sample were separately digested using PCT, and each analyzed by SWATH-MS in duplicates. The distribution of CV values of protein quantification for each digestion batch, injection batch, and total variation is shown as violin plots where the kernel density curves shape the violin, the white node in the center denotes median value, and the black box inside the violin shows the same interquantile range as the box-and-whiskers plot. (b) Bar plot of average median CV of injection, digestion and the entire workflow (Total).

Mentions: Next, we assessed variation among technical triplicates for each of the 4 digests. We determined the CV of triplicate injections of the 4 digests in three batches and plotted the CV distribution as violin plots (Fig. 3a). The 12% values missing in at least one map were not considered in this analysis. The median CV for repeated MS injection batches was between 10% and 12%. The variation of PCT-assisted digestion across the samples was slightly higher, with the median rising to 16-20%. Median CV of all 12 replicates (Total) was 24% (Fig. 3b). This is the overall median technical variation expected for the same tissue sample measured using the entire PCT-SWATH workflow. This value is comparable to that achieved in our laboratory by S/MRM in a label-free quantification mode if applied at high throughput to the model organism Saccharomyces cerevisiae 20. It is worth noting that excluding the 12% missing values may artificially influence the CV values, particularly among replicates. However, the abundance of proteins did not have significant influence on the CV (Supplementary Fig. 2).


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)

Reproducibility of PCT-SWATH(a) Four aliquots of a human kidney tissue sample were separately digested using PCT, and each analyzed by SWATH-MS in duplicates. The distribution of CV values of protein quantification for each digestion batch, injection batch, and total variation is shown as violin plots where the kernel density curves shape the violin, the white node in the center denotes median value, and the black box inside the violin shows the same interquantile range as the box-and-whiskers plot. (b) Bar plot of average median CV of injection, digestion and the entire workflow (Total).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4390165&req=5

Figure 3: Reproducibility of PCT-SWATH(a) Four aliquots of a human kidney tissue sample were separately digested using PCT, and each analyzed by SWATH-MS in duplicates. The distribution of CV values of protein quantification for each digestion batch, injection batch, and total variation is shown as violin plots where the kernel density curves shape the violin, the white node in the center denotes median value, and the black box inside the violin shows the same interquantile range as the box-and-whiskers plot. (b) Bar plot of average median CV of injection, digestion and the entire workflow (Total).
Mentions: Next, we assessed variation among technical triplicates for each of the 4 digests. We determined the CV of triplicate injections of the 4 digests in three batches and plotted the CV distribution as violin plots (Fig. 3a). The 12% values missing in at least one map were not considered in this analysis. The median CV for repeated MS injection batches was between 10% and 12%. The variation of PCT-assisted digestion across the samples was slightly higher, with the median rising to 16-20%. Median CV of all 12 replicates (Total) was 24% (Fig. 3b). This is the overall median technical variation expected for the same tissue sample measured using the entire PCT-SWATH workflow. This value is comparable to that achieved in our laboratory by S/MRM in a label-free quantification mode if applied at high throughput to the model organism Saccharomyces cerevisiae 20. It is worth noting that excluding the 12% missing values may artificially influence the CV values, particularly among replicates. However, the abundance of proteins did not have significant influence on the CV (Supplementary Fig. 2).

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.The measured proteins clearly distinguished tumorous kidney tissues from healthy tissues and differentiated distinct histomorphological kidney cancer subtypes.

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