Identification and quantification of protein species using 1DE and 2DE

processing was the same, decreasing the technical variation.

The examination of proteins identified in 1DE-r1 and 2DE revealed a number of proteins for which a number of protein species were present. In order to examine the protein species, protein coverage was shown using the position in the protein of the first amino acid of each identified peptide. The pI and the MW of each protein species were calculated using the protein sequence that was spanned by the identified peptides. The experimental pI and MW for each slice was calculated as the median of the proteins that show maximum intensity (Figure 7) in this row/column. Eight proteins that showed presence of multiple species were selected and then the separation/identification of those species was compared between the 1DE and the 2DE separation. The selected proteins were vimentin (VIM), Figure 16 and Figure 17, plastin 3 (PLS3), Figure 18 and Figure 19, T-complex protein 1 subunit alpha (TCP1), Figure 20 and Figure 21, lamin-B1 (LMNB1), Figure 22 and Figure 23, lamin A/C (LMNA), Figure 24 and Figure 25. In addition, three proteins showed characteristic horizontal spread in the 2DE approach and different ratio H/L ratios. These proteins were LMNA,

Figure 26 and Figure 27, F-actin-capping protein subunit beta (CAPZB), Figure 28 and Figure 29 and UPF0568 protein C14orf166 (C14orf166), Figure 30 and Figure 31. A comparison of the separation of the different species of each of those proteins in the 1DE and 2DE approaches was performed.

Figure 16. Identification of protein species of vimentin (VIM), 2DE experiment.

A) Distribution of protein intensity of VIM. Gel slices with bold borders were selected to display the ratio H/L of the identified peptides and the protein sequence coverage. B) Sequence coverage, presented as the position of the first amino acid of each identified peptide, versus ratio H/L of the identified peptides. Experimental pI and MW vs theoretical pI and MW of the protein species: column 2 / row 18: pI 5.3 / 5.05, MW 60 / 54; column 1 / row 25:

pI 5.27 / 4.7, MW 49 / 43; column 22 / row 43: pI 8.41 / 6.51, MW 27 / 27. Color scheme based on peptide PEP (red: PEP <1E-11, yellow: PEP<0.0001, light blue: PEP < 0.05, dark blue:

PEP>0.05).

Figure 17. Identification of protein species of vimentin (VIM), 1DE experiment.

A) Distribution of protein MS intensity of VIM. Gel slices with bold borders were selected to display the ratio H/L of the identified peptides and the protein sequence coverage. B) Sequence coverage, presented as the position of the first amino acid of each identified peptide, versus ratio H/L of the identified peptides. Color scheme based on peptide PEP (red:

PEP <1E-11, yellow: PEP<0.0001, light blue: PEP < 0.05, dark blue: PEP>0.05).

Figure 18. Identification of protein species of plastin 3 (PLS3), 2DE experiment.

A) Distribution of protein MS intensity of PLS3. Gel slices with bold borders were selected to display the ratio H/L of the identified peptides and the protein sequence coverage. B) Sequence coverage, presented as the position of the first amino acid of each identified peptide, versus ratio H/L of the identified peptides. Experimental pI and MW vs theoretical pI and MW of the protein species: column 6 / row 16: pI 5.62 / 5.83, MW 65 / 66; column 8 / row 20: pI 5.95 / 5.94, MW 59 / 56. Color scheme based on peptide PEP (red: PEP <1E-11, yellow:

PEP<0.0001, light blue: PEP < 0.05, dark blue: PEP>0.05).

Figure 19. Identification of protein species of plastin 3 (PLS3), 1DE experiment.

A) Distribution of protein MS intensity of PLS3. Gel slices with bold borders were selected to display the ratio H/L of the identified peptides and the protein sequence coverage. B) Sequence coverage, presented as the position of the first amino acid of each identified peptide, versus ratio H/L of the identified peptides. Color scheme based on peptide PEP (red:

PEP <1E-11, yellow: PEP<0.0001, light blue: PEP < 0.05, dark blue: PEP>0.05).

Figure 20. Identification of protein species of T-complex protein 1 subunit alpha (TCP1), 2DE experiment.

A) Distribution of protein MS intensity of TCP1. Gel slices with bold borders were selected to display the ratio H/L of the identified peptides and the protein sequence coverage. B) Sequence coverage, presented as the position of the first amino acid of each identified peptide, versus ratio H/L of the identified peptides. Experimental pI and MW vs theoretical pI and MW of the protein species: column 9 / row 18: pI 6.11 / 5.8, MW 60 / 60; column 8 / row 30:

pI 5.9 / 5.67, MW 40 / 28; column 12 / row 43: pI 6.4 / 5.68, MW 26.8 / 26. Color scheme based on peptide PEP (red: PEP <1E-11, yellow: PEP<0.0001, light blue: PEP < 0.05, dark blue:

PEP>0.05).

Figure 21. Identification of protein species of T-complex protein 1 subunit alpha (TCP1), 1DE experiment.

A) Distribution of protein MS intensity of TCP1. Gel slices with bold borders were selected and evaluated for ratio H/L and sequence coverage. B) Sequence coverage, presented as the position of the first amino acid of each identified peptide, versus ratio H/L of the identified peptides. Color scheme based on peptide PEP (red: PEP <1E-11, yellow: PEP<0.0001, light blue: PEP < 0.05, dark blue: PEP>0.05).

Figure 22. Identification of protein species of lamin-B1 (LMNB1), 2DE experiment.

A) Distribution of protein MS intensity of LMNB1. Gel slices with bold borders were selected and evaluated for ratio H/L and sequence coverage. B) Sequence coverage, presented as the position of the first amino acid of each identified peptide, versus ratio H/L of the identified peptides. Experimental pI and MW vs theoretical pI and MW of the protein species: column 3 / row 16: pI 5.4 / 5.1, MW 65 / 66; column 3 / row 25: pI 5.2 / 5.07, MW 49 / 44. Color scheme based on peptide PEP (red: PEP <1E-11, yellow: PEP<0.0001, light blue: PEP < 0.05, dark blue: PEP>0.05).

Figure 23. Identification of protein species of lamin-B1 (LMNB1), 2DE experiment.

A) Distribution of protein MS intensity of LMNB1. Gel slices with bold borders were selected and evaluated for ratio H/L and sequence coverage. B) Sequence coverage, presented as the position of the first amino acid of each identified peptide, versus ratio H/L of the identified peptides. Color scheme based on peptide PEP (red: PEP <1E-11, yellow: PEP<0.0001, light blue: PEP < 0.05, dark blue: PEP>0.05).

Figure 24. Identification of protein species of lamin A/C (LMNA), 2DE experiment.

A) Distribution of protein MS intensity of LMNA. Gel slices with bold borders were selected to display the ratio H/L of the identified peptides and the protein sequence coverage. B) Sequence coverage, presented as the position of the first amino acid of each identified peptide, versus ratio H/L of the identified peptides. Experimental pI and MW vs theoretical pI and MW of the protein species: column 13 / row 17: pI 6.3 / 8.3, MW 134 / 62; column 8 / row 26 (protein sequence: 0 – 377): pI 5.9 / 5.8, MW 49 / 43; column 9 / row 34 (protein sequence 0 – 261): pI 6.1 / 5.9, MW 40 / 30. Color scheme based on peptide PEP (red: PEP <1E-11, yellow:

PEP<0.0001, light blue: PEP < 0.05, dark blue: PEP>0.05).

Figure 25. Identification of protein species of lamin A/C (LMNA), 1DE experiment.

A) Distribution of protein MS intensity of LMNA. Gel slices with bold borders were selected and evaluated for ratio H/L and sequence coverage. B) Sequence coverage, presented as the position of the first amino acid of each identified peptide, versus ratio H/L of the identified peptides. Color scheme based on peptide PEP (red: PEP <1E-11, yellow: PEP<0.0001, light blue: PEP < 0.05, dark blue: PEP>0.05).

Figure 26. Identification of protein species of lamin A/C (LMNA), 2DE experiment.

A) Distribution of protein MS intensity of LMNA. B) Distribution of protein ratio H/L of LMNA.

Gel slices with bold borders were selected to display the ratio H/L of the identified peptides and the protein sequence coverage. C) Sequence coverage, presented as the position of the first amino acid of each identified peptide, versus ratio H/L of the identified peptides. Color scheme based on peptide PEP (red: PEP <1E-11, yellow: PEP<0.0001, light blue: PEP < 0.05, dark blue: PEP>0.05).

Figure 27. Identification of protein species of lamin A/C (LMNA), 1DE experiment.

A) Distribution of protein MS intensity of LMNA. Gel slices with bold borders were selected to display the ratio H/L of the identified peptides and the protein sequence coverage. B) Sequence coverage, presented as the position of the first amino acid of each identified peptide, versus ratio H/L of the identified peptides. Color scheme based on peptide PEP (red:

PEP <1E-11, yellow: PEP<0.0001, light blue: PEP < 0.05, dark blue: PEP>0.05).

Figure 28. Identification of protein species of F-actin-capping protein subunit beta (CAPZB), 2DE experiment.

A) Distribution of protein MS intensity of CAPZB. B) Distribution of protein ratio H/L of CAPZB. Gel slices with bold borders were selected to display the ratio H/L of the identified peptides and the protein sequence coverage. C) Sequence coverage, presented as the position of the first amino acid of each identified peptide, versus ratio H/L of the identified peptides. Color scheme based on peptide PEP (red: PEP <1E-11, yellow: PEP<0.0001, light blue: PEP < 0.05, dark blue: PEP>0.05).

Figure 29. Identification of protein species of F-actin-capping protein subunit beta (CAPZB), 1DE experiment.

A) Distribution of protein MS intensity of CAPZB Gel slices with bold borders were selected to display the ratio H/L of the identified peptides and the protein sequence coverage. B) Sequence coverage, presented as the position of the first amino acid of each identified peptide, versus ratio H/L of the identified peptides. Color scheme based on peptide PEP (red:

PEP <1E-11, yellow: PEP<0.0001, light blue: PEP < 0.05, dark blue: PEP>0.05).

Figure 30. Identification of protein species of UPF0568 protein C14orf166 (C14orf166), 2DE experiment.

A) Distribution of protein MS intensity of C14orf166. B) Distribution of protein ratio H/L of CAPZB. Gel slices with bold borders were selected to display the ratio H/L of the identified peptides and the protein sequence coverage. C) Sequence coverage, presented as the position of the first amino acid of each identified peptide, versus ratio H/L of the identified peptides. Color scheme based on peptide PEP (red: PEP <1E-11, yellow: PEP<0.0001, light blue: PEP < 0.05, dark blue: PEP>0.05).

Figure 31. Identification of protein species of UPF0568 protein C14orf166 (C14orf166), 1DE experiment.

A) Distribution of protein MS intensity of C14orf166. Gel slices with bold borders were selected to display the ratio H/L of the identified peptides and the protein sequence coverage. B) Sequence coverage, presented as the position of the first amino acid of each identified peptide, versus ratio H/L of the identified peptides. Color scheme based on peptide PEP (red: PEP <1E-11, yellow: PEP<0.0001, light blue: PEP < 0.05, dark blue: PEP>0.05).

An evaluation of the distribution of the intensity and the ratio H/L of protein species revealed distinct patterns of distribution over the 2DE gel. Interestingly there was an agreement between the experimental pI and MW of the 2DE slice and the theoretical pI and MW of the potential protein species. Examples of good agreement were proteins PLS3 (Figure 18) and LMNB1 (Figure 22). Protein VIM showed good agreement in the MW values while the pI values showed differences of between 0.5 and 1 (Figure 16). Protein TCP1 was also a prominent example with species showing similar experimental and theoretical pI values. Based on the sequence coverage in the different fractions, the protein seemed to be split into N and C terminal part (Figure 20) and both protein species had almost the same ratio H/L value of -1.96 on log2 scale. These results show that multiple protein species per protein indeed exist. For several proteins, the identified species showed different H/L ratios. For example the full length VIM species had a median ratio of 2.43 while the species missing the 100 N-terminal amino acids had a median ratio of – 0.65 (log2 scale). The full length PLS3 protein species had a ratio of 1.57 while the protein species lacking the N-terminal 125 amino acids had a ratio of -2.72. The full length of LMNB1 had a ratio of -0.3 and the second protein species, which lacked approximately 200 amino acids on the C terminus, showed a ratio of -2.35. These results indicate that while multiple species of a protein are present in the protein sample, they can have different relative abundance between CPC-2D and CPC-3D. For the selected set of proteins that showed the presence of multiple species, the performance, in terms of separation and identification, of the 1DE analysis was evaluated. Interestingly the 1DE performed worse than the 2DE analysis. In several cases protein separation by 1DE did not provide enough evidence for the presence of multiple protein species per protein. In the cases where the presence of more than one protein species was indicated, there was carryover from other slices suggesting insufficient separation power. An example for this is protein VIM. For protein VIM there is evidence for the presence of three protein species (Figure 16) on the 2DE gel, one of which lacks 100 amino acids at the N-terminus. The 1DE analysis also revealed the presence of two species, with the second species possibly also lacking 100 amino acids at the N-terminus. This could be inferred from the slightly higher ratio H/L of the peptides from the N-terminus region relative to the rest of the protein. Nevertheless, the evidence for the existence of a second protein species was much less conclusive in the 1DE analysis compared to the 2DE analysis. For three other proteins: PLS3 (Figure 19), TCP1 (Figure 21) and LMNB1 (Figure 23), there was no conclusive evidence for the existence of more than one protein species. The 2DE analysis revealed the presence of at least three proteins species of LMNA (Figure 24) while the 1DE analysis indicated the existence only of two protein species (Figure 25). These results strongly suggest that 2DE

is capable of efficiently separating/isolating different species of the same protein, which aids their confident identification and characterization.

Protein separation by pI is the crucial difference between 1DE and 2DE. In order to evaluate the benefit of this additional separation dimension, three proteins that showed different horizontal ratio H/L distribution were selected and their identification in the 1DE analysis was examined. One prominent example of the presence of several protein species, which also show different relative abundance between CPC-2D and CPC-3D, was protein LMNA (

Figure 26). For LMNA a distinct horizontal spread on the 2DE gel of the protein identification and decreasing ratio H/L from right to the left was observed. An evaluation of three slices from the horizontal spread revealed that in all three slices the full-length protein was present. This indicated that the spread is most probably a result of a modification of the protein, which affected its pI and thus horizontal migration on the 2DE gel, and, furthermore, that the degree of modification of the protein was different in CPC-2D and CPC-3D resulting in different ratios across the horizontal spread. Another interesting observation related to LMNA is that the full-length protein has a theoretical pI and MW, which are very different from the experimental pI and MW of the 2DE slice (Figure 24). The theoretical pI and MW of full length LMNA is 8.3 and 62 KDa. The highest intensity of the full length LMNA protein is in column 13, row 17, which corresponds to pI of 6.3 and MW of 134 KDa. This again indicates that this is a modified version of the protein which shifts its pI to more acidic and its MW to higher values. One candidate for such a modification is phosphorylation (Zhu, Zhao et al. 2005). As expected, the 1DE analysis revealed the presence of the full length LMNA protein (Figure 27) but due to the absence of separation by pI no additional species at the same MW were revealed.

The analysis of two other proteins: CAPZB (Figure 28) and c14orf166 (Figure 30) also revealed the presence of protein species that have the same MW but are separated based on their pI. Interestingly, in both examples the ratio H/L decreased from right to left across the 2DE gel. Similarly to the example of LMNA, the 1DE analysis of both proteins (Figure 29, Figure 31) did not reveal the presence and differential quantification of separate protein species. These results indicate that due to its separation power, the 2DE approach allows for the identification and quantification of multiple protein species. In comparison, the identification rate of protein species was much lower when using the 1DE approach.

3.4 Establishment of three dimensional PAGE-pIEF-LC-MS/MS