Comparative analysis of the BBM SDS gel bands 2, 9 and 11

The quality and the reproducibility of the BBM preparation was assessed at the precursor ion level on the basis of the analysis of the selected gel bands 2, 9 and 11. The goal of this analysis was two-fold: firstly, to explore whether a differential analysis based on precursor mass intensities would align with the findings derived from the comparison of the same samples based on protein identification; and secondly, to evaluate to which extent the (ir)reproducibility of the LC-MS system could be observed in a more complex protein mixture such as used for the BBM identification study.

As already mentioned in section 4.5.2, all comparisons were performed using three representative SDS-PAGE bands, Bands 2, 9, and 11. Band 2 represented a very abundant, very well defined high molecular mass band (expectation: excellent reproducibility) while Band 9 represented a diffuse, very faint band (expectation: not so good reproducibility). Band 11 also displayed a well defined band but differed from Band 2 by the fact that the proteins present in the mass range of the gel might also encompass proteolytic products (expectation:

average reproducibility). For simplicity reason, only representative scatter plots of chosen sample comparisons will be shown. The complete set of scatter plots and Spearman correlation values for all comparisons can be found in appendix B3.

Fig. 4.31 shows the comparison scatter plots for the three injection replicates of the band 2.

The three replicates were very reproducible according to the scatter plot representation of all common MS signals and of the MS signals that correspond to successful MS/MS measurement. The Spearman correlation values for all comparisons were above 0.98 underlining the very good linearity of the signals at the diagonal. Similarly to what was observed with the simple Dionex protein mixture, the MS signals which were successfully assigned a peptide sequence through MS/MS analysis were in large of high signal intensity (panel B). However, this ion population represented less than 10% of the overall common MS signals (compare N between panel A and B).

Figure 4.31: Scatter plot representations of the injection replicates of the band 2. Panel A:

Scatter plots of all the common MS signals between the replicates. Panel B: Scatter plots of the MS signals that correspond to successful MS/MS measurements. N is the number of common signals. The Spearman value reflects the similarity of the signal intensities between the compared samples (ideal case Spearman correlation=1). Each red dot line parallel to the diagonal represents a two fold difference, i.e. across three lines, the fold change is 2*2*2=8.

Accordingly, the axes represent an arbitrary mass spectrometric intensity (in counts) in log2 units. All sample denominations are as described in Fig. 4.21.

Further, the comparison scatter plots for a triplicate analysis of the band 2 excised from three adjacent SDS-PAGE lanes is shown in Fig. 4.32. The added variability of this technical step compared to the triplicate injection replicate was rather moderate as judged by the uniform and only slightly lower Spearman correlation values compared to the injection replicates. The main feature differentiating those two analyses were a broadening of the ion distribution along the diagonal, especially noticeable at the lower ion intensity scale, and a ion correlation matrix that did not include the least intense ion, probably because of lack of reproducibility (compare Fig. 4.31 and Fig. 4.32 panels A and panels B side by side). Interestingly, within the triplicate analysis included in Fig. 4.32, the samples pool_wBBM_2_2 and pool_wBBM_3_2 appeared to correlate slightly better to each other than with the sample pool_wBBM_2_1, a finding that was not observed from the Venn diagrams analysis of the same samples (compare with Fig. 4.22, panel B).

Spearman: 0.99 Spearman: 0.988 Spearman: 0.985

Spearman: 0.996 Spearman: 0.995 Spearman: 0.993

N = 1337 N = 1337 N = 1337

A

B

N = 17420 N = 17420 N = 17420

Injection replicates for band 2

Pool_wBBM_1_2_a

Spearman: 0.99 Spearman: 0.988 Spearman: 0.985

Spearman: 0.996 Spearman: 0.995 Spearman: 0.993

N = 1337 N = 1337 N = 1337

A

B

N = 17420 N = 17420 N = 17420

Injection replicates for band 2

Figure 4.32: Scatter plot representations of the variability for the band 2, cut horizontally from adjacent identical lanes. Panel A: Scatter plots of all the common MS signals between the replicates. Panel B: Scatter plots of the MS signals that correspond to successful MS/MS measurements. N is the number of common signals. The Spearman value reflects the similarity of the signal intensities between the compared samples (ideal case Spearman correlation=1). Each red dot line parallel to the diagonal represents a two fold difference, i.e.

across three lines, the fold change is 2*2*2=8. Accordingly, the axes represent an arbitrary mass spectrometric intensity (in counts) in log2 units. All sample denominations are as described in Fig. 4.21.

Similarly, the comparison scatter plots for a triplicate analysis of the band 2 excised from a lane run in three separate SDS-PAGE gels is shown in Fig. 4.33. As expected from the previous analysis, the added variability of this technical step was very comparable to the variability obtained from the triplicate bands excised from a single SDS-PAGE gel. The measurement reproducibility depended mostly on how accurately the bands were defined and excised from the SDS-PAGE gel, the type (wide/narrow; defined/diffuse, etc.) of bands that was analyzed, and how identically the gels to be compared had run. Interestingly, in this triplicate analysis shown in Fig. 4.33, the samples pool_wBBM_A2 and pool_wBBM_B2 appeared to correlate slightly better to each other than with the samples pool_wBBM_C2, a finding that was not observed from the Venn diagrams analysis of the same samples (compare with Fig. 4.22, panel C). Taken together, the results obtained in Fig. 4.32 and 4.33 suggest that the SDS-PAGE step appeared non-critical for the reproducibility of an experiment as long as the parameters for protein separation and band excision were kept tightly controlled.

A

Spearman: 0.907 Spearman: 0.916 Spearman: 0.887

Spearman: 0.893 Spearman: 0.955 Spearman: 0.86

N = 1337 N = 1337 N = 1337

N = 17420 N = 17420 N = 17420

Same gel variation for band 2

SameGel_pool_wBBM_1_2

Spearman: 0.907 Spearman: 0.916 Spearman: 0.887

Spearman: 0.893 Spearman: 0.955 Spearman: 0.86

N = 1337 N = 1337 N = 1337

N = 17420 N = 17420 N = 17420

Same gel variation for band 2

Figure 4.33: Scatter plot representations of the band 2 from identical BBM samples, loaded in three different gels. Panel A: Scatter plots of all the common MS signals between the replicates. Panel B: Scatter plots of the MS signals that correspond to successful MS/MS measurements. N is the number of common signals. The Spearman value reflects the similarity of the signal intensities between the compared samples (ideal case Spearman correlation=1). Each red dot line parallel to the diagonal represents a two fold difference, i.e.

across three lines, the fold change is 2*2*2=8. Accordingly, the axes represent an arbitrary mass spectrometric intensity (in counts) in log2 units. All sample denominations are as described in Fig. 4.21.

The comparison scatter plots for the LC-MS signals of the band 2 originating from three different technical BBM preparations is shown in Fig. 4.34. Since all technical steps (samples run on the same SDS-PAGE gel, band excision, digestion, column and buffer batch, LC-MS conditions) were maintained constant, the observed variability for the common signals should mainly reflect the reproducibility of the BBM preparation. The common MS signals (Panel A) and the common MS signals that were assigned to a successful MS/MS identification (Panel B) were clustered at the diagonal indicating a high level of similarity between the three BBM preparations. This observation was supported by a high Spearman correlation value (above 0.94) for all the comparisons. This high degree of reproducibility was also noted for the preparation variation analysis of bands 9 and 11 (results not shown, for more details see appendix B) and correlated with the findings obtained from the protein identification level (see Fig. 4.22, panel E, Fig. 4.23, panel E and Fig. 4.24, panel D). These results demonstrate

Difgel_pool_wBBM_C_2

Difgel_pool_wBBM_B_2 Difgel_pool_wBBM_B_2 Difgel_pool_wBBM_C_2

Spearman: 0.854 Spearman: 0.945 Spearman: 0841

Spearman: 0817 Spearman: 0.964 Spearman: 0.814

N = 1337 N = 1337 N = 1337

N = 17420 N = 17420 N = 17420

Different gel variation for band 2

Difgel_pool_wBBM_A_2 Difgel_pool_wBBM_A_2

Spearman: 0.854 Spearman: 0.945 Spearman: 0841

Spearman: 0817 Spearman: 0.964 Spearman: 0.814

N = 1337 N = 1337 N = 1337

N = 17420 N = 17420 N = 17420

Different gel variation for band 2

Difgel_pool_wBBM_A_2 Difgel_pool_wBBM_A_2

Difgel_pool_wBBM_B_2 Difgel_pool_wBBM_B_2 Difgel_pool_wBBM_C_2

Difgel_pool_wBBM_C_2 Difgel_pool_wBBM_A_2 Difgel_pool_wBBM_A_2

A

B

that the BBM preparation protocol could be performed in a very stable and reproducible manner that added only a minimal variability compared to other technical steps.

Figure 4.34: Scatter plot representations of the preparation variability for the band 2 from three BBM technical replicate preparations. Panel A: Scatter plots of all the common MS signals between the replicates. Panel B: Scatter plots of the MS signals that correspond to successful MS/MS measurements. N is the number of common signals. The Spearman value reflects the similarity of the signal intensities between the compared samples (ideal case Spearman correlation=1). Each red dot line parallel to the diagonal represents a two fold difference, i.e. across three lines, the fold change is 2*2*2=8. Accordingly, the axes represent an arbitrary mass spectrometric intensity (in counts) in log2 units. All sample denominations are as described in Fig. 4.21.

Finally, the comparison scatter plots for the LC-MS signals of the band 2, 9 and 11 originating from three different technical BBM preparations is shown in Fig. 4.35, 4.36 and 4.37, respectively. In this last set of analyses, however, the technical steps were not as tightly controlled and the samples were analyzed in random orders.

According to the scatter plot representation and the Spearman correlation values (Fig. 4.35), the variability observed for the band 2 was considerably lower than for the bands 9 and 11. In this experiment, variability must originate from the technical steps since the three BBM preparation were found to be highly reproducible in the previous section. In our experience, most

Spearman: 0.964 Spearman: 0.960 Spearman: 0.941

Spearman: 0.971 Spearman: 0.960 Spearman: 0.956

N = 1337 N = 1337 N = 1337

N = 17420 N = 17420 N = 17420

Preparation variation for band 2

PV_wBBM_2_2 PV_wBBM_3_2

Spearman: 0.964 Spearman: 0.960 Spearman: 0.941

Spearman: 0.971 Spearman: 0.960 Spearman: 0.956

N = 1337 N = 1337 N = 1337

N = 17420 N = 17420 N = 17420

Preparation variation for band 2

PV_wBBM_2_2 PV_wBBM_3_2

PV_wBBM_2_2 PV_wBBM_3_2 PV_wBBM_1_2

PV_wBBM_3_2

of the technical variability was due to different column history, column and LC buffer changes

Figure 4.35: Scatter plot representations of the total variability for the band 2 from three BBM technical replicate preparations, randomly analyzed. Panel A: Scatter plots of all the common MS signals between the replicates. Panel B: Scatter plots of the MS signals that correspond to successful MS/MS measurements. N is the number of common signals. The Spearman value reflects the similarity of the signal intensities between the compared samples (ideal case Spearman correlation=1). Each red dot line parallel to the diagonal represents a two fold difference, i.e. across three lines, the fold change is 2*2*2=8. Accordingly, the axes represent an arbitrary mass spectrometric intensity (in counts) in log2 units. All sample denominations are as described in Fig. 4.21.

Indeed, the triplicates of the band 2 were randomly analyzed along the whole experiment but the column and LC buffer remained the same for the three analyses. The Spearman correlation value was slightly lower than was found for the preparation variation alone but remained in the same range. The slightly elevated variability could be due to the manner the gel bands were excised from the gel (one after the other versus horizontal parallel cutting) and to the LC column history. Thus, according to the Spearman correlation value, the samples TV_wBBM_3_2 and TV_wBBM_2_2 were significantly better correlated than with TV_wBBM_1_2 probably, because these two first samples were analyzed very closely to each other at the LC-MS level (only 2 samples separated the two band 2 replicates).

The variability observed for band 9 was significantly higher than that found for band 2 and was reflected in the scatter plots and the Spearman correlation values (Fig. 4.36). In our experience, this large increase in variability was due to column and buffer changes during the

analysis of the band 9 triplicates. Thus, the correlation of the replicates TV_wBBB_1_9 and TV_wBBM_3_9 was much higher than with TV_wBBM_2_9. TV_wBBB_1_9 and TV_wBBM_3_9 were analyzed on two different columns but using the same LC buffer batch, while TV_wBBM_2_9 was analyzed using a different column and another LC buffer batch.

More details about the analysis sequence of all the samples included in this experiment can be found in the process variation measurement times in the appendix B.

Figure 4.36: Scatter plot representations of the total variability for the band 9 from three BBM technical replicate preparations, randomly analyzed. Panel A: Scatter plots of all the common MS signals between the replicates. Panel B: Scatter plots of the MS signals that correspond to successful MS/MS measurements. N is the number of common signals. The Spearman value reflects the similarity of the signal intensities between the compared samples (ideal case Spearman correlation=1). Each red dot line parallel to the diagonal represents a two fold difference, i.e. across three lines, the fold change is 2*2*2=8. Accordingly, the axes represent an arbitrary mass spectrometric intensity (in counts) in log2 units. All sample denominations are as described in Fig. 4.21.

Column and buffer changes affected the analysis of band 11 in a similar fashion as seen in band 9 (see Fig. 4.37). Thus, the two replicates TV_wBBM_2_11 and TV_wBBM_3_11 clustered significantly better to each other than with the replicate TV_wBBM_1_11. Indeed, the replicates TV_wBBM_2_11 and TV_wBBM_3_11 were measured using different columns but the same LC solvent batch, while TV_wBBM_1_11 was analyzed using a different column and another LC buffer batch.

Interestingly, for all bands considered (but mostly for bands 9 and 11), the significantly lower comparability of the total variability analysis compared to the preparation variability analysis

Total variation for band 9 A Total variation for band 9

A

was only partially observed at the protein identification level (see Fig. 4. 22, panel D and E;

Fig. 4.23, panels D and E; and Fig. 4.24, panels C and D). The ion correlation matrix which

Figure 4.37: Scatter plot representations of the total variability for the band 11 from three BBM technical replicate preparations, randomly analyzed. Panel A: Scatter plots of all the common MS signals between the replicates. Panel B: Scatter plots of the MS signals that correspond to successful MS/MS measurements. N is the number of common signals. The Spearman value reflects the similarity of the signal intensities between the compared samples (ideal case Spearman correlation=1). Each red dot line parallel to the diagonal represents a two fold difference, i.e. across three lines, the fold change is 2*2*2=8. Accordingly, the axes represent an arbitrary mass spectrometric intensity (in counts) in log2 units. All sample denominations are as described in Fig. 4.21.

was used to create the scatter plots depends heavily on a reproducible RT and m/z parameters linked with the measured ion signal intensity, whose variability was minimized by measuring the samples to compare immediately one after another. Moreover, the correlation matrix took into account all MS signals that were commonly assigned to the samples, with a majority of them being rather of low intensity and subject to higher variability. In contrast, sample comparison by protein identification was based on a MS signal that was successfully assigned to an amino acid sequence through a MS/MS identification. Since this ion population comprised mostly well behaved, reproducible high intensity MS signal, the correlation matrix illustrated through Venn diagrams was much more resistant to intensity variability than for the common MS signals.

Total variation for band 11 A Total variation for band 11

A

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