Table 1.
Identified proteins from Daphnia pulex acclimated to 10°C or 20°C
| Spot no. | Specificity N10: N20 | Matched peptide sequences | Sequence coveragea) | Mascot scoreb) | Mrgel/Mrpredicted | pI gel/pI predicted | SP Length | PP Length | Putative function (symbolic name) |
| Proteolytic enzymes | |||||||||
| 36 | 0.3* | 1. VVAGEHSLR 2. SVDVPVVDDDTCNR |
8.9% | 149 | 30/26–29 | 4.4/4.4–4.8 | 15 | 27 | Trypsin (TRY5L) |
| 1. LTAAEEPTRVEIR 2. IRNDVALIK |
7.5% | 80 | 30/25–30 | 4.4/4.5–5.3 | 18 | 48 | Chymotrypsin (CHY1A) | ||
| 37 | 0.2* | 1. GVTDLTIFR 2. VVAGEHSLR 3. VVAGEHSLRTDSGLEQNR |
9.8% | 159 | 29/26–29 | 4.4/4.4–4.8 | 15 | 27 | Trypsin (TRY5F) |
| 1. VVAGEHSLR 2. SVDVPVVDDDTCNR |
8.9% | 149 | 29/26–29 | 4.4/4.4–4.8 | 15 | 27 | Trypsin (TRY5L) | ||
| 38 | 0.5* | 1. GLADADIAVFK 2. LIWMGQYNR 3. YYRDELAGK |
10.7% | 123 | 29/30 | 4.5/4.5 | 19 | Endoribonuclease-like protein (ERNA) | |
| 1. GLADADIAVFK 2. LIWMGQYNR 3. YYRDELAGK |
8.0% | 123 | 29/39 | 4.5/4.6 | 20 | Endoribonuclease-like protein (ERNB) | |||
| 1. VVAGEHSLR 2. SVDVPVVDDDTCNR |
8.9% | 149 | 29/26–29 | 4.5/4.4–4.8 | 15 | 27 | Trypsin (TRY5L) | ||
| 1. GVTDLTIFR 2. VVAGEHSLR |
6.5% | 80 | 29/26–29 | 4.5/4.4–4.8 | 15 | 27 | Trypsin (TRY5F) | ||
| 39 | 0.4* | 1. VVAGEHSLR 2. SVDVPVVDDDTCNR |
8.9% | 149 | 29/26–29 | 4.6/4.4–4.8 | 15 | 27 | Trypsin (TRY5L) |
| 1. GVTDLTIFR 2. VVAGEHSLR |
6.5% | 120 | 29/26–29 | 4.6/4.4–4.8 | 15 | 27 | Trypsin (TRY5F) | ||
| 40 | 0.6* | 1. TTEEYYVSVQK 2. TGGGCYSYIGR |
6.5% | 112 | 25/23–27 | 4.5/4.7–4.6 | ? | 39 ? | Astacin (ACN2) |
| 1. GVTDLTIFR 2. VVAGEHSLR |
6.5% | 109 | 25/26–29 | 4.5/4.4–4.8 | 15 | 27 | Trypsin (TRY5F) | ||
| 41 | 0.4* | 1. LTAAEEPTR 2. LTAAEEPTRVEVR 3. IINDVALIK |
9.1% | 141 | 25/25–30 | 4.7/4.4–5.0 | 18 | 47 | Chymotrypsin (CHY1C) |
| 28 | 1.2 | see [12] | Peptidase M13 Peptidase M2 Carboxylesterase, type B Sphingomyelin phosphodiesterase Sphingomyelin phosphodiesterase |
||||||
| 31 | 0.6 | see [12] | 30/34–45 30/35–46 29/26–29 |
4.8/4.9–4.8 4.8/5.1–4.9 4.4/4.4–4.8 |
16 16 15 |
92 93 27 |
Carboxypeptidase A (CPA1A) Carboxypeptidase A (CPA1B) Trypsin (TRY5F) |
||
| 32 | 0.3 | see [12] | 23/24–27 | 5.0/5.2–5.4 | 17 | 24 | Trypsin (TRY4B) | ||
| Egg yolk proteins & precursors | |||||||||
| 43 | 7.3* | see Figure 2 | /190–220 | /6.4–6.7 | 17–20 | Vitellogenin (VTG1, VTG2, VTG4) | |||
| 44 | 7.1* | see Figure 3 see Figure 2 |
15.7% 2.8% |
271 | 25/42 25/220 |
5.8/5.3 5.8/6.7 |
17 17 |
Actin Vitellogenin (VTG1) |
|
| 45 | 5.9* | see Figure 2 | 2.2% | 132 | 21/190 | 5.2/6.4 | 20 | Vitellogenin (VTG4) | |
| 46 | 5.2* | see Figure 2 | 2.9% | 361 | 21/220 | 5.9/6.7 | 17 | Vitellogenin (VTG1) | |
| 47 | 4.9* | see Figure 3 see Figure 2 |
9.6% 2.0% |
25/42 25/220 |
5.6/5.3 5.6/6.7 |
17 | Actin Vitellogenin (VTG1, VTG2) |
||
| 1. EDQMDYLEEK 2. LLVEKER 3. YSVDEELNK |
3.6% | 25/83 | 5.6/4.7 | HSP90 | |||||
| 49 | 4.4* | see Figure 2 | ?.?% | ??? | 21/190–220 | 4.8/6.4–6.7 | 17–20 | Vitellogenin (VTG1, VTG2, VTG4) | |
| 50 | 4.2* | see Figure 2 | 2.2% | 163 | 20/220 | 5.7/6.7 | 17 | Vitellogenin (VTG1) | |
| 52 | 3.7 | see Figure 2 | 3.0% | 344 | 18/220 | 5.1/6.7 | 17 | Vitellogenin (VTG1, VTG2) | |
| Cytoskeleton & muscle proteins | |||||||||
| 48 | 4.5* | see Figure 2 see Figure 3 |
?.?% | ??? | 27/42 | 5.2/5.3 | Actin Vitellogenin (VTG4) |
||
| 1. EQLDEESEAK 2. AEELEDAKR 3. ATVLANQMEK |
27/220 | 5.2/5.9 | Myosin heavy chain (MHC-1) | ||||||
| 1. LTTDPAFLEK 2. NAAAVHEIR 3. GDLGIEIPPEK |
27/?? | 5.2/? | Pyruvate kinase | ||||||
| 51 | 3.6* | see Figure 3 | ?.?% | ??? | 36/42 | 5.7/5.3 | Actin | ||
| ATPase | |||||||||
| 42 | 9.8* | 2. GNEDLSTAILK 3. MDELQLFK 4. GDIFIVR 5. KQLALIK 6. EMVELPLR |
5.1% | 214 | 16/89 | 5.3/5.0 | AAA+ ATPase | ||
| Carbohydrate-modifying enzymes | |||||||||
| 35 | 1.2 | see [12] | α-Amylase (AMY) | ||||||
| 34 | 1.0 | see [12] | Exo-β-1,3-Glucanase (EXG5) | ||||||
| 1 | 0.4 | see [12] | Cellubiohydrolase (CEL7A) | ||||||
| 29 | 0.3 | see [12] | Endo-β-1,4-Glucanase (CEL9A) Paramyosin (PMY) |
||||||
| 30 | 0.6 | see [12] | Endo-β-1,4-Mannanase (MAN5A) β-1,3-Glucan-binding protein |
||||||
| 19 | 0.6 | see [12] | Enolase (ENO) | ||||||
Identification was based on 2D gel electrophoresis and nano-HPLC-ESI-MS/MS analysis of trypsin-digested proteins matched against the "Frozen Gene Catalog" of the D. pulex protein database [2]. The compiled information includes the spot number (Figure 1A, B), the 10-to-20°C expression ratio, the number and sequences of matched peptides, the sequence coverage, the Mascot score as a statistical measure of identification probability, the experimental and theoretical molecular weight (Mr) and isolectric point (pI) of the mature protein (without signal peptide), the predicted length of the N-terminal signal peptide (SP) in secretory proteins, as well as the putative function and symbolic name of the protein. The length of the putative pro-peptide (PP) is additionally provided for proteolytic enzymes that are secrected as inactive precursors (zymogens). The predicted Mr and pI values of zymogens and the mature enzymes are given as value ranges. The amino acid sequences of the identified proteins were derived from the gene models listed in Table 2. a)percentage of predicted protein sequence covered by matched peptides. b)Probability-based MOWSE score: -10*Log(P), where P is the probability that the observed match is a random event. Scores >38 indicate identity or extensive homology (p < 0.05). Protein scores are derived from ions scores as a non-probabilistic basis for ranking protein hits. The Mascot-score calculation was performed using whole-protein sequence (including the N-terminal signal peptide in case of extracellular proteins). *p < 0.05 (t-Test).