Regardless of the method used to determine total protein concentration as shown in Figure , there is a large and rapid influx of proteins (within 0.5 hour) into AH after both types of incision with a return to baseline after 12 hours for clear corneal incision and after 24 hours for limbal incision. The limbal incision resulted in a greater increase in protein concentrations compared to clear corneal incision at all time points with highest concentration differences of about 3.2-fold, 2.7-fold and 9.4-fold at 0.5, 2 and 12 hour time points, respectively.
Additional file 1
, Figure S1 shows representative 2-DE gel images for AH samples taken after clear corneal incision (panel A) and after limbal incision (Additional file 2
, Figure S1, panel B) at 5 time points with pre-surgery AH sample (0 hour). Left column represents low protein amount loaded into gels for individual time points (decrease concentration; 50-78 μg of total protein) and right column shows gels with high protein amount loaded (increase concentration; 280-376 μg of total protein) for corresponding time points with enlargements showing areas exhibiting highest concentration of protein spots. Unfortunately, the same experiments could not be performed at high protein load for AH samples with low protein concentration (pre-surgery samples, and samples at time points 12, 24 and 48 hours for clear corneal incision (Additional file 1
, Figure S1, panel A) and samples at time points 24 and 48 hours for limbal incision (Additional file 2
, Figure S1, panel B)), due to a limitation in a loading volume for 1st
dimension of 2-DE (maximum of 350 μL for 18 cm long strip, see section Methods for more details) and due to a limited amount of AH sample available (approx. 0.2 mL per rabbit). Although the obvious solution of the former problem seems to be the use of concentrating, buffer exchange or centrifugal filtration, we finally opted to use originally collected samples to keep and treat all AH samples the same way. For example, AH contains other components in addition to proteins and using the buffer exchange method would change AH original composition. As well, the spot quantity comparisons between samples would be more complicated if concentration method is used, and using the centrifugal filtration may cause undesirable protein loss.
Additional file 3
, Figure S2 shows representative 2-DE gel images with high protein loads of AH samples (280-376 μg of total protein) obtained from clear corneal incision (panel A; 0.5 and 2 hour time points) and limbal incision (panel B; 0.5, 2 and 12 hour time points), 2 different animals per time point are shown.
dimension of 2-DE, the corresponding volume of AH sample was used to ensure that the similar amount of total protein was loaded into the gels at low concentration (Additional files 1
, Figure S1, panels A and B) or at high concentration (Additional file 3
, Figure S2). In order to carry out a quantitative comparison among gels, AH concentration loaded per gel was kept similar, which meant different volumes of the AH were used.
The list of detected proteins is given in Table (the highest number of peptides in spot for corresponding protein are listed if found in multiple gel spot(s)). For proteins detected by 1 peptide, peptide amino sequence and peptide charge are included in Table and they are included only if identified in multiple gel spots, meaning either present in i) different gel spots of appropriate surgery type (clear corneal and/or limbal incision samples), ii) identical spots over different time points within the same type of incision, iii) identical spots but for different type of incision (clear corneal vs limbal), and iv) identical spots within the same type of incision but for different protein loads (low vs high loads).
List of unique proteins identified in AH surgery samples.
Based on 2-DE gel image analysis results (Additional file 3
, Figure S2), the protein spots that were up- or down-regulated were calculated and Figure shows the examples of the numbers for protein spot changes from 0.5 to 2 hour time points within clear corneal and limbal incision procedures (panel A; fold change > ± 1.5 and p < 0.05; 2 rabbits per time point) and between clear corneal and limbal incisions at 0.5 and 2 hour time points (panel B; fold change > ± 1.5 and p < 0.05; 2 rabbits per time point). As can be seen, from total 1343 protein spots detected for protein spot changes from 0.5 to 2 hour time points within clear corneal incision (panel A, left Venn diagram), only 17 and 14 protein spots, respectively, matched our criteria for up- and down-regulation, and the numbers are similar in magnitude for all four comparisons presented in Figure . The examples of proteins identified in several protein spots with fold changes and p-values are listed in Tables and for protein spot changes from 0.5 to 2 hour time point within clear corneal and limbal incision procedures and for protein spot changes between clear corneal and limbal incisions at 0.5 and 2 hour time points, respectively. Proteins included in Table (but not listed in Tables an ) have been identified either at time points different from 0.5 and 2 hours or in low concentration 2-DE gels (see Additional files 1
, Figure S1, panels A and B). Additional file 4
, Figure S3 visualizes the protein spots which changed from 0.5 to 2 hour time point within clear corneal (panel A) and limbal incision (panel B) procedures and between clear corneal and limbal incisions at 2 hour time point (panel C) as detected in 2-DE gels. The proteins identified in individual 2-DE spots can be found in Tables and . Additional file 4
, Figure S3 shows the zoomed areas for each gel spot to allow easier comparison between corresponding time points and types of incision.
Figure 2 Number of protein spot changes. A - from 0.5 to 2 hour time points within clear corneal (left diagram) and limbal (right diagram) incision procedure; B - between clear corneal and limbal incision procedures at 0.5 hour (left diagram) and 2 hour (right (more ...)
Protein spot changes from 0.5 hour to 2.0 hour time points within clear corneal (A) and limbal (B) incision procedures.
Protein spot changes between clear corneal and limbal incisions at 0.5 hour (A) and 2 hour (B) time points.
Figure shows the distribution of 80 identified unique proteins. In panel A, 16 and 25 proteins were identified in clear corneal and limbal incision samples, respectively, and 39 proteins were common to both types of surgery (see Table , 4th
column). Protein database search and literature search revealed that 62.5% and 36.3% were cellular and serum proteins, respectively, location was not determined for 1.2% of proteins (Figure , panel B and Table , 3rd
column). Compared to our previous study (Ref. [19
]), in which we listed the proteins detected in healthy AH of rabbits, only 26 proteins have been common to both our studies, whereas 54 proteins (67.5%) were newly detected here in surgery AH samples (Figure , panel C and Table , 5th
Figure 3 Distribution of 80 unique proteins. A - proteins identified in clear corneal and limbal incision AH samples; B - cellular, serum and origin not determined proteins; C - proteins already identified in healthy AH (previous Ref ) and proteins newly found (more ...)
Proteins found in AH surgery samples and specific only for one type surgery (either clear corneal or limbal incision) but not identified in healthy AH (Ref [19
]) were: mucin 16, DEAD box polypeptide 55, programmed cell death 8, tRNA wybutosine-synthesizing protein 2, cytochrome P450, E2F transcription factor 4, tubulin, collagen type I, eukaryotic translation elongation factor 1, stratifin, gelsolin, valosin-containing protein, sterol O-acyltransferase 1, peroxisomal membrane protein 2, isocitrate dehydrogenase 3, olfactory receptor Olr1474, methylmalonyl-CoA mutase, limbin, AMMECR1, rabconnectin-3, aristaless 3, lumican, annexin A1, complement component 8 and crystallin lambda 1. Majority of these proteins are cellular proteins, (88%; based on Protein Knowledgebase (UniProtKB/Swiss-Prot), Gene Ontology database and literature search), only two of them are serum proteins (lumican and complement component 8). For example, 5 cellular proteins, i.e. tubulin, collagen type I, eukaryotic translation elongation factor 1, stratifin (also called 14-3-3 protein sigma) and gelsolin, were exclusively present in AH samples undergoing limbal incision.
Proteins identified in the identical protein spots of samples collected over various time points (corresponding protein spots are incised from different time point gels) were either serum proteins - serum albumin, apolipoprotein A-I, alpha-1-antiproteinase F, transferrin, Try 10-like trypsinogen, paraoxonase, alpha-2-HS-glycoprotein, transhyretin or cellular proteins - SLAM family member 9, tudor domain-containing protein 12, iodotyrosine dehalogenase 1 protein, cytochrome P450, and peroxisomal membrane protein 2. The proteins were present in AH during various period of time after surgery, e.g. SLAM family member 9 was found in samples collected at 0.5, 2 and 12 hours after both clear corneal and limbal incision, tudor domain-containing protein 12 and cytochrome P450 in samples after limbal incision at time points 0.5, 2, 12 and 24 hours and 12 and 24 hours, respectively, and paraoxonase and peroxisomal membrane protein 2 were present in samples collected at 0.5 and 2 hours following clear corneal incisison.
To obtain more extensive proteome coverage, 2-DE with high protein load were performed for AH samples collected at time points 0.5-2 hours (clear corneal incision) and 0.5-12 hours (limbal incision), respectively (Additional file 3
: Figure S2). We identified additional 14 proteins (not identified in healthy AH samples) which were only detected with high but not with low protein load.