Limits...
The Impact II, a Very High-Resolution Quadrupole Time-of-Flight Instrument (QTOF) for Deep Shotgun Proteomics.

Beck S, Michalski A, Raether O, Lubeck M, Kaspar S, Goedecke N, Baessmann C, Hornburg D, Meier F, Paron I, Kulak NA, Cox J, Mann M - Mol. Cell Proteomics (2015)

Bottom Line: The new reflectron and detector improve resolving power compared with the previous model up to 80%, i.e. to 40,000 at m/z 1222.We analyzed the ion current from the inlet capillary and found very high transmission (>80%) up to the collision cell.Finally, after high pH reversed-phase fractionation we identified 9515 proteins in a triplicate measurement of HeLa peptide mixture and 11,257 proteins in single measurements of cerebellum-the highest proteome coverage reported with a QTOF instrument so far.

View Article: PubMed Central - PubMed

Affiliation: From the ‡Proteomics and Signal Transduction, Max-Planck-Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany;

No MeSH data available.


Related in: MedlinePlus

A, Correlation of single shot LC-MS/MS measurements of haploid (h) and diploid (d) yeast samples. All technical replicate correlation values were > 0.98. B, Quantitative differences between the haploid and diploid yeast proteome. Proteins marked in red are significantly more abundant in haploid cells. C, Principal component analysis (PCA) of LFQ protein expression levels in mouse hepatoma (Hepa 1–6), mouse embryonic fibroblasts (MEF) and motoneuronal cell lines (NSC-34). D, Loading of the first two principal components as obtained from the PCA plotted in C. The inset indicates significantly enriched annotations along the PC axes (FDR < 0.05). The respective enrichment scores are specified in parentheses.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4587313&req=5

Figure 6: A, Correlation of single shot LC-MS/MS measurements of haploid (h) and diploid (d) yeast samples. All technical replicate correlation values were > 0.98. B, Quantitative differences between the haploid and diploid yeast proteome. Proteins marked in red are significantly more abundant in haploid cells. C, Principal component analysis (PCA) of LFQ protein expression levels in mouse hepatoma (Hepa 1–6), mouse embryonic fibroblasts (MEF) and motoneuronal cell lines (NSC-34). D, Loading of the first two principal components as obtained from the PCA plotted in C. The inset indicates significantly enriched annotations along the PC axes (FDR < 0.05). The respective enrichment scores are specified in parentheses.

Mentions: To test the workflow in a systems biology context, we analyzed proteome changes of diploid and haploid (Matα cell) S. cerevisiae. We analyzed 2 μg of yeast digest from haploid and diploid cells in technical quadruplicates with our standard 90 min gradient. This identified 3769 proteins using “match between runs.” For statistical analysis, we only considered LFQ intensities that were detected in at least three replicates of the haploid or diploid groups. After filtering, 3222 proteins remained for further analysis (Experimental Procedures). Remaining missing values were imputed from a normal distribution. The technical replicates correlated much more with each other than they correlated to the other genotype (R2 greater 0.98 versus R2 about 0.92; Fig. 6A). As in our previous large-scale analysis on SILAC labeled haploid and diploid yeast (50), we found transposons more abundant in haploid cells than in diploid cells (Fig. 6B). Ste3, the pheromone a factor receptor, was specific to haploid yeast, as expected from its mating status. Also absent in diploid but present in haploid cells were Sst2, a GTPase-activating protein for Gpa1 (51), which, consistently, showed higher expression in haploid cells. Conversely, Sps100, which is a sporulation-specific wall maturation protein turned out to be specific for diploid cells. Doing such systems-wide comparisons by traditional methods would have required thousands of individual Western blots. Even compared with our previous large-scale study performed by quantitative MS (50), we here used less than 1% of yeast input material and measurement time. This illustrates the rapidity by which MS-based proteomics is becoming a viable method for answering biological questions.


The Impact II, a Very High-Resolution Quadrupole Time-of-Flight Instrument (QTOF) for Deep Shotgun Proteomics.

Beck S, Michalski A, Raether O, Lubeck M, Kaspar S, Goedecke N, Baessmann C, Hornburg D, Meier F, Paron I, Kulak NA, Cox J, Mann M - Mol. Cell Proteomics (2015)

A, Correlation of single shot LC-MS/MS measurements of haploid (h) and diploid (d) yeast samples. All technical replicate correlation values were > 0.98. B, Quantitative differences between the haploid and diploid yeast proteome. Proteins marked in red are significantly more abundant in haploid cells. C, Principal component analysis (PCA) of LFQ protein expression levels in mouse hepatoma (Hepa 1–6), mouse embryonic fibroblasts (MEF) and motoneuronal cell lines (NSC-34). D, Loading of the first two principal components as obtained from the PCA plotted in C. The inset indicates significantly enriched annotations along the PC axes (FDR < 0.05). The respective enrichment scores are specified in parentheses.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4587313&req=5

Figure 6: A, Correlation of single shot LC-MS/MS measurements of haploid (h) and diploid (d) yeast samples. All technical replicate correlation values were > 0.98. B, Quantitative differences between the haploid and diploid yeast proteome. Proteins marked in red are significantly more abundant in haploid cells. C, Principal component analysis (PCA) of LFQ protein expression levels in mouse hepatoma (Hepa 1–6), mouse embryonic fibroblasts (MEF) and motoneuronal cell lines (NSC-34). D, Loading of the first two principal components as obtained from the PCA plotted in C. The inset indicates significantly enriched annotations along the PC axes (FDR < 0.05). The respective enrichment scores are specified in parentheses.
Mentions: To test the workflow in a systems biology context, we analyzed proteome changes of diploid and haploid (Matα cell) S. cerevisiae. We analyzed 2 μg of yeast digest from haploid and diploid cells in technical quadruplicates with our standard 90 min gradient. This identified 3769 proteins using “match between runs.” For statistical analysis, we only considered LFQ intensities that were detected in at least three replicates of the haploid or diploid groups. After filtering, 3222 proteins remained for further analysis (Experimental Procedures). Remaining missing values were imputed from a normal distribution. The technical replicates correlated much more with each other than they correlated to the other genotype (R2 greater 0.98 versus R2 about 0.92; Fig. 6A). As in our previous large-scale analysis on SILAC labeled haploid and diploid yeast (50), we found transposons more abundant in haploid cells than in diploid cells (Fig. 6B). Ste3, the pheromone a factor receptor, was specific to haploid yeast, as expected from its mating status. Also absent in diploid but present in haploid cells were Sst2, a GTPase-activating protein for Gpa1 (51), which, consistently, showed higher expression in haploid cells. Conversely, Sps100, which is a sporulation-specific wall maturation protein turned out to be specific for diploid cells. Doing such systems-wide comparisons by traditional methods would have required thousands of individual Western blots. Even compared with our previous large-scale study performed by quantitative MS (50), we here used less than 1% of yeast input material and measurement time. This illustrates the rapidity by which MS-based proteomics is becoming a viable method for answering biological questions.

Bottom Line: The new reflectron and detector improve resolving power compared with the previous model up to 80%, i.e. to 40,000 at m/z 1222.We analyzed the ion current from the inlet capillary and found very high transmission (>80%) up to the collision cell.Finally, after high pH reversed-phase fractionation we identified 9515 proteins in a triplicate measurement of HeLa peptide mixture and 11,257 proteins in single measurements of cerebellum-the highest proteome coverage reported with a QTOF instrument so far.

View Article: PubMed Central - PubMed

Affiliation: From the ‡Proteomics and Signal Transduction, Max-Planck-Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany;

No MeSH data available.


Related in: MedlinePlus