Chromatographic separation and enrichment of target peptides

In the first chromatographic dimension, the peptides are separated based on their hydrodynamic radius by size-exclusion chromatography, whereas in the second dimension they are separated depending on their hydrophobicity by reversed phase chromatography at neutral pH (Figure 14). At

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both steps, all the fractions obtained are screened by mass spectrometry, and only the ones containing the target peptides are processed further. At this stage, the signal from the error-containing peptides is still too low to be detected, so that only the respective heavy-labeled standards are monitored. For the subsequent quantification the peptides are loaded onto a nanoflow chromatographic system and separated by reverse phase at acidic pH prior to their detection in the mass spectrometer. Although the last two dimensions both rely on reversed phase chromatography, the different pH at which they are performed confer separation power, especially for those peptides which contain residues whose protonation status changes upon pH shift – i.e., aspartate and glutamate – and whose separation can be altered by using different pH range. Because our peptides normally contain one ofthese amino acids, their chromatographic behavior changes from neutral to acidic pH ensuring their efficient separation. For an initial screening of error frequencies, we have chosen to follow the Arg to His substitutions (Table 4), known to be relatively abundant (Zhang et al., 2013). In addition, this type of amino acid misincorporation is particularly favourable to be investigated using our enrichment protocols because it changes the tryptic pattern of the protein which we proteolyzed using trypsin (which cleaves at R and K residues). The proteolysis of the error-containing protein yields longer peptides that elute earlier from the size-exclusion column than the products of digestion of the correct protein which are shorter. Thereby, the complexity of the sample and the background noise are efficiently reduced. Low-abundance erroneous peptides are enriched, separated within each other and from the more abundant cognates allowing us to increase the column load, pulling low abundance-peptides in the dynamic range of the instrument so that their signal can be detected.

Figure 14. Distribution of peptide elution intervals in the first two chromatographic dimensions. EF-Tu peptides displaying different chemical property can be efficiently separated according to their size and hydrophobicity. Separated peptides are represented by the red dots. The size of each dot reflects the number of fractions in which the respective peptide is eluting.

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Table 4. General properties of R to H substituted peptides. Distinct physicochemical characteristics allow for efficient separation and correct identification of error-containing peptides. Hydrophobicity

factors are estimated using the peptide analyzer tool

https://www.thermofisher.com/de/de/home/life-science/protein-biology/peptides-proteins/custom-peptide-synthesis-services/peptide-analyzing-tool.html.

Misincorporation Peptide Length Hydrophobicity

R45H TYGGAAHAFDQIDNAPEEK 19 28.03

R59H AHGITINTSHVEYDTPTR 18 24.83

R231H GTVVTGHVER 10 13.43

R234H VEHGIIK 7 13.53

R270H LLDEGHAGENVGVLLR 16 32.49

R280H AGENVGVLLHGIK 13 27.51

R284H HEEIER 6 5.86

R319H DEGGHHTPFFK 11 19.94

R328H GYHPQFYFR 9 28.31

R378H FAIHEGGR 8 13.16

R382H EGGHTVGAGVVAK 13 15.52

In some cases, enrichment and analysis of error-containing peptides which have very similar physicochemical characteristics to their cognate peptides (e.g., FESEVYILSK peptide (Table 5)) might not be achieved in the first two dimensions (Figure 15). However, the separation of target peptides from the highly abundant cognate peptide is essential, as without their separation the sample complexity cannot be reduced. To improve the separation, further chromatographic steps are required which are tailored to the characteristics of specific peptides. We chose to add the third chromatographic step, reversed phase at acidic pH prior to the reversed phase performed on the nanoflow system. Although three rounds of reversed phase might not be fully orthogonal, they synergistically provide not only a further reduction of sample complexity, but also improve the separation of histidine-containing peptides due to the different pH. The second reversed phase chromatography, therefore, enhances the separation of error-containing peptides from the correct FESEVYILSK, before that the sample is loaded on the LC-MS/MS system (Figure 16). This reduces local interferences in SRM quantification and allows to load more target peptide without saturating the LC system, both improving the signal to noise ratio and signal intensity.

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Table 5. Physicochemical characteristics of peptides differing by a few amino acids may be very similar. When compared to the cognate peptide FESEVYILSK, peptide variants with one, two or three amino acid difference have very similar charachteristics in terms of both length and hydrophobicity.

Hydrophobicity factor has been estimated using the peptide analyzer tool available on the website of Thermo Fischer Scientific at the link https://www.thermofisher.com/de/de/home/life- science/protein-biology/peptides-proteins/custom-peptide-synthesis-services/peptide-analyzing-tool.html

Errors Peptide Length Hydrophobicity

Cognate FESEVYILSK 10 28.2

F305L LESEVYILSK 10 26.41

E306D FDSEVYILSK 10 28.51

E308D FESDVYILSK 10 27.45

Y310H FESEVHILSK 10 23.69

Y310N FESEVNILSK 10 25.23

E306D-E308D FDSDVYILSK 10 27.83

E306D-Y310H FDSEVHILSK 10 24.00

E308D-Y310H FESDVHILSK 10 23.10

E306D-Y310N FDSEVNILSK 10 25.56

E308D-Y310N FESDVNILSK 10 24.64

E306D-E308D-Y310H FDSDVHILSK 10 23.41

Figure 15. Poor separation of similar peptides in the first two chromatographic dimensions.The two-dimensional separation of peptides that differ in only few amino acids with respect to the correct peptide is poor. Separated peptides are represented by dots (error-containing peptides in red, correct FESEVYILSK in blue). The size of each dot reflects the number of fractions in which they are eluting.

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Figure 16. Elution pattern of similar peptides in reversed phase chromatography runs at neutral and acidic pH.The different pH of the mobile phase changes the separation profiles of peptides which contain ionizing amino acids. Separated peptides are represented by dots (error-containing peptides in red, correct FESEVYILSK in blue). The size of each dot reflects the number of fractions in which peptides elute.

2.2Identification of enriched error-containing peptides