3.3 Mass spectrometry
3.3.1 Comparison of proteins secreted from different stages
LC-MS/MS was also carried out on whole worm extracts to confirm differences in protein composition compared to E/S products as suggested by 1-D gel electrophoresis. In a preliminary test a “whole worm” analysis was performed to test whether it is possible to detect proteins with relatively low amounts of individuals. For this method larvae were counted and picked indivi-dually and subjected to mass spectrometric analysis, as described in 2.3.3.10. 1–1,000 iL3 were counted, 1-D SDS PAGE was performed and the bands were analysed (Figure 3.3.1-1).
Looking at the number of identified proteins in the respective batches a clear increase can be seen. The maximum number of 150 proteins was identified in the last batch with 1,000 larvae, representing a volume of approximately 0.011 mm3. Due to the effort it takes to prepare the worms and considering that cellular components, membrane proteins and proteins of low abun-dance might be underrepresented using this approach, it was decided to use the regular native worm extracts for the comparative analysis with E/S products.
By applying LC-MS/MS for the E/S products and extracts of iL3, pf and fls it was possible to identify 1,081 proteins in total. The Venn diagram shows the distribution of identified proteins in the E/S products (Fig 3.3.1-2) and the extracts (Fig 3.3.1-3) within the three stages. For the E/S products 587 proteins have been found in all the stages. 195 proteins, representing roughly one third of the total protein count of all stages, have been found solely in the iL3. 78 proteins (13 %) have been found in the parasitic females and 34 proteins (5 %) have been found in the free-living stages only, whereas 140 proteins (23 %) have been found in samples from every stage.
Figure 3.3.1-1 1-D SDS PAGE of whole iL3s after heating in loading buffer. Seven different amounts of larvae, shown by the numbers above the gel, have been counted and loaded onto the gel. The boxes indicate the gel pieces that have been cut. The table on the right side shows the number of proteins that have been identified in the respec-tive batch.
NO iL3 NO proteins
1 1
5 2
15 5
50 29
150 65
500 119
1,000 150 Number of iL3
191 kDa 14 kDa
S 1 5 15 50 150 500 1,000
995 proteins have been identified in the extracts of iL3, pf and fls. Here the fls represent the largest number with 723 proteins (73 %) followed by the pf with 574 proteins (58 %) and the iL3 with 489 proteins (49 %). 282 proteins (28 %) have been found only in the fls and almost the same amount of 271 proteins (27 %) in all stages. The lowest number of proteins could be allo-cated in the iL3 only. Here 68 proteins (9 %) were found and 125 proteins (13 %) were found in the pf only.
195
140
19 100
78
21
34 iL3 (454)
fls (214) pf
(339)
Figure 3.3.1-2 Schematic representation of proteomic data. The Venn diagram shows the distribution of identified S. ratti E/S products over three life cycle stages – iL3, pf, and fls. Numbers in brackets show the numbers of identified proteins in each stage in total.
pf (574)
iL3 (489)
fls (723) 271
71
99
282 79
68
125
Figure 3.3.1-3 Schematic representation of proteomic data. The Venn diagram shows the distribution of identified S. ratti extract proteins of the three life cycle stages – iL3, pf, and fls. Numbers in brackets show the numbers of identified proteins in each stage in total.
The graph in figure 3.3.1-4 the E/S products and in the extracts
found for the E/S products and the extracts it can be seen that there is a large shift from the iL3 to the fls. The fls have a low number of secreted proteins compared to the proteins present in the extract. Whereas the iL3 have a high number of secreted proteins as in the
extract proteins. The number of identified proteins in all stages shows only a small increase in the extract whereas the number of proteins found in iL3 and pf is smaller.
The graph in figure 3.3.1-5
E/S products and extracts. It should be stated that the stage with most prote in the E/S products is the iL3 followed by the pf and the fls. The fls most proteins found in the extracts
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
E/S
Figure 3.3.1-4 Schematic representation of identified proteins within the percent the composition of identified proteins in the E/S
single stage or combination of stages in which proteins were found
4 shows in percent the composition of the identified proteins in products and in the extracts for the various stages. By direct comparison of the results products and the extracts it can be seen that there is a large shift from the iL3 The fls have a low number of secreted proteins compared to the proteins present in the extract. Whereas the iL3 have a high number of secreted proteins as in the combined iL3 and pf The number of identified proteins in all stages shows only a small increase in the extract whereas the number of proteins found in iL3 and pf is smaller.
5 shows the compilation of unique and common proteins in the products and extracts. It should be stated that the stage with most proteins being found only
products is the iL3 followed by the pf and the fls. The fls, however, most proteins found in the extracts, followed by the pf and the iL3.
Extract
Schematic representation of identified proteins within the different stages. The graph shows the composition of identified proteins in the E/S products and the extracts. The different colours show the single stage or combination of stages in which proteins were found.
identified proteins in the various stages. By direct comparison of the results products and the extracts it can be seen that there is a large shift from the iL3 The fls have a low number of secreted proteins compared to the proteins present in the combined iL3 and pf The number of identified proteins in all stages shows only a small increase in
common proteins in the ins being found only is the stage with
iL3, pf and fls fls
pf and fls pf iL3 and fls iL3 and pf iL3
stages. The graph shows in products and the extracts. The different colours show the
3.3.2 Abundant proteins in E/S