Determinación de causas potenciales

Data reduction step

When using intensities to estimate relative protein abundances, it is important to make sure that we are not inflating measurements with data from instrument or chemical noise.

Just as the TIC varies dramatically from scan to scan, even within a single salt pulse, the intensity and number of noise peaks in each MS/MS scan also change. Each spectrum has different distributions of peak intensities and therefore different noise peak cutoffs, so we looked for the inflexion points where the bottom-ranked peak intensities indicated strong linearity. Due to the high variability of individual scans, these noise levels ranged widely in their absolute values (between 5-300 peaks and 1000-5000 summed intensities due to

noise), On average, this noise-reducing method resulted in removing about 80% of the peaks and roughly 20% of the TIC, suggesting superior performance that neither intensity

Figure 3.3). However, further examination revealed that the data reduction step was not noticeably improving the matched ion intensities for a given peptide, so this method was not used in the final quantification analyses.

Scan-based metrics

Each scan averaged an MIT of 2.87e5 with a standard deviation of 1.34e6, accounting for 22% (+/- 12%) of the TIC. Whereas the TICs ranged from 1.29e4 to 7.07e8, the MITs ranged from 1.95e3 to 2.64e8. MITs were not correlated with the TICs, so the matching process was demonstrated to be an informative calculation step. Similarly, the MITs were not correlated with the number of peaks (or quality peaks) in a scan, so they could not have been substituted by those quality metrics. Each salt pulse contributed a slightly different distribution of scans- as noted by the distribution of TICs and the average MITs. Figure 3.4 below also illustrates how the number and range of matched ion intensities of each salt pulse differed for a single run. Almost a 50:50 split between the number of b and y ions were observed. For each scan, an average of 11.78 and 11.99 b and y ions were matched, contributing an average of 4.8e4 and 1.25e5 to a scan’s MIT, respectively. On average, 24 peaks (+/- 9) matched within a scan, predominately reflecting the number

Figure 3.3. Validating the use of matched ion intensities instead of other simple features inherent to MS/MS scans.

(A) Each MS/MS scan’s TIC was compared to the number of fragment peaks within the scan to see if there was a correlation.

(B) For each peptide-spectrum match that passed the typical filtering criteria, the MS/MS scan’s TIC was compared to the matched ion intensity to see whether the matched ion intensity was a consistent fraction of the TIC.

(B) (A)

of matched peaks from the most abundant charge states (+2 and +3), which matched 22.5 and 23.45 peaks per scan. The +1 scans averaged 15 matching peaks and the +4 scans averaged 53.8 peaks, differing from the other scans’ metrics primarily due to their relatively increased and decreased number of possible peaks matched.

Peptide-based metrics

In keeping with the NSAF assumption that more opportunities to sample an analyte would increase its abundance, MIT measurements were compared to peptide length in order to assess whether there was a correlation. Similarly, peptides with a higher charge state have more opportunities to generate fragment ions, so the correlation between MITs and charge state (and number of possible fragment ions). PSM-level MITs grouped into peptide MITs were not biased for more opportunities based on any of these metrics. As Figure 3.5 suggests, peptide MITs were, however, different between charge states and salt pulses. The distribution of MITs for a highly abundant peptide, TVIEVLVENGNVSK (700 total SpC and 2.45e8 MIT) is illustrated in Figure 3.4. The average of the MITs collected for each salt pulse are slightly different for the exact same analyte. The shift downwards (smaller intensities) with each consecutive salt pulse reveals that the peptide is continuing to be measured even amidst growing competition for identification. Looking at any of these salt pulses individually would be a misrepresentation of the peptide’s behavior across the entire run. Even looking at the cumulative distribution of MITs observed for this analyte does not completely capture the behavior of this peptide sequence. As the graph in Figure 3.5 suggests, this peptide behaves like quite different analytes depending on its charge state- perhaps just as differently as two peptide sequences altogether. If one is trying to validate the distribution of the peptide’s MITs as a component of the protein’s abundance within a run, it is more accurate to compare the peptide’s distribution across technical replicates than it is to compare two peptides from the same protein within a single run.

Figure 3.6A highlights the similarities in MIT distributions between the same peptide identified in two technical replicates. To determine whether the peptide distribution

Figure 3.4. The distributions of an abundant peptide’s matched ion intensity for each of the 11 salt pulses in a single run.

0.00E+00 2.00E+05 4.00E+05 6.00E+05 8.00E+05 1.00E+06 1.20E+06 0 2 4 6 8 10 12 Ma tch ed Io n Int ens it y Salt pulse Peptide Matched Ion Intensity

would behave the same across different loading amounts, the same sample was loaded on to a column in 2 different concentrations (25 μg and 67 μg). Figure 3.6B graphs how the peptide MIT distribution follows the same shape and general trend between the two concentrations and systematically reflects the expected shift in intensities between the two runs. Therefore, peptide MITs are considered reproducible across replicates and across loading amounts. However, not all peptides were identified in all replicate measurements. When the peptides are assembled into protein measurements, these inconsistencies in identification warrant careful consideration to either filter or normalize for the disparities.

Figure 3.5. Abundant peptide demonstrates different matched ion intensity distributions depending on its charge state.

(A) The same peptide sequence was captured by vastly different SpC throughout a single run. The +2 species was observed over 700 times, compared to the 3-60 SpC detected by the other species. (B) An inset of the graph above to illustrate that the carbamylated (N- terminus + 43) species of +1 and its non-modified form followed the same general trend, as did the +3 modified and non-modified species.

-20 0 20 40 60 80 100 120 140 1 3 5 7 9 Counts

Log10 Matched Ion Intensity

Peptide Matched Ion Intensity Distribution by Analyte

"+1 No mod" +1 Mod + 2 Mod + 3 No mod + 3 Mod -2 0 2 4 6 8 10 12 14 16 18 0 2 4 6 8 Counts

Log10 Matched Ion Intensity

Peptide Matched Ion Intensity Distribution by Analyte

"+1 No mod" +1 Mod "+ 3 mod" + 3 no Mod (B) (A)

Figure 3.6. Peptide matched ion intensities are reproducible.

(A) Peptide matched ion intensities are consistent across technical replicates. (B) Peptide matched ion intensities may reflect the relative differences in the amount of sample analyzed by MS. 0 50 100 150 200

1.00E+05 1.00E+06 1.00E+07

Counts

Matched Ion Intensity

Peptide Matched Ion Intensity Distributions Between Replicates

0 50 100 150 200 250 6.00E+04 9.60E+05 Counts

Matched Ion Intensity

Peptide Matched Ion Intensity Distributions Within Different Loading Amounts

25ug Load 67ug Load

(B) (A)

3.2 Augmented and Refined Peptide Identifications from