Two-dimensional gel electrophoresis (2D-PAGE)

Liver and pancreas tissue samples were taken from three wt and three KO mice. Tissue samples were dissolved in lysis buffer consisting of 9 M urea, 2% DTT, 2% Triton X-100 and 2 % IPG buffer. Horizontal isoelectric focusing was performed using a non-linear pH3-10NL IPG strip, rehydrated for 20 hours at room temperature with a rehydration buffer (8 M urea, 2 % CHAPS, 2% IPG-buffer, 0,3% DDT). The first dimension was carried out on a Multiphor ΙΙ Electrophoresis unit at 500 V for 5 hours and at 3500 V for 14.5 hours. Prior to the second dimension, the IPG strip was equilibrated twice with an equilibration buffer (0.05 M Tris-base, 6 M Urea, 26% glycerol, 1 % SDS), and transferred to a polyacrylamide gel. The second dimension separation was run vertically at 50 V for 19 hours [8]. One gel was performed for each tissue sample from one mouse, thus three biological replicates were analysed for each tissue.

Silver staining

Visualisation was achieved by sliver staining, suitable for quantitative protein analysis [8,9]. Briefly, gels where fixed overnight in a fixation solution (50% ethanol, 12 % acetic acid and 0.0185% formaldehyde), then washed three times for 20 minutes in 35% ethanol, pre-treated for one min in 0.02 % Na₂S₂O₃, and rinsed in water 3 times for 2-3 min. Staining of gels with silver nitrate was preformed for 20 minuets, after which they again where rinsed twice with water. Gels where developed using a developer solution (6% Na₂CO₃, 0.0185% formaldehyde, 0.0004% Na₂S₂O₃, 5 H₂O) for approximately 3 min. Development where finally stopped in a stop solution (40% ethanol, 12% acetic acid). The gels where dried in cellophane sheets and sealed in plastic bags.

Image analysis

Silver stained gels where scanned using an ImageQuant LAS-4000 (GE Healthcare) and TIFF images of gels where imported into the PDQuest software analysis program. Two proteomic analyses where done; one with pancreatic tissue and one with hepatic tissue. The 2D-gels where divided in two groups; one representing wt mice and another representing KO mice. Protein spots where automatically defined and adjusted to the background. The quality of each protein spot was critically evaluated in order to ensure that all relevant protein spots were identified. The pixel-intensity of each protein spot was translated to a proportional protein volume, which was normalized to the total density of the gel and given in parts per million, ppm. Finally, re-analysis was done manually on all matched spots. Differentially expressed spots were defined as spots that differed at least 2-fold in average relative volume between the groups of wt and KO mice. The results where considered to be significant when p<0.05 using a students t-test. These spots where selected for further identification by LC-MS/MS.

Protein identification

Protein identification by LC-MS/MS was performed essentially as previously described [8]. Briefly, gels containing protein spots selected for identification were re-hydrated in water, the cellophane sheets were peeled off and the protein spots were excised from the gels. Proteins were in-gel digested with trypsin. Gel pieces were first dehydrated in acetonitrile, then dried and the proteins reduced for 1h at 56°C in 10 mM dithiotreitol (DTT) and 100 mM NH₄HCO₃. The solution was exchanged with 55 mM iodoacetamide in 100 mM NH₄HCO₃ for 45 min. Then the gel pieces were washed in 100 mM NH₄HCO₃, dehydrated in acetonitrile, rehydrated in 100 mM NH₄HCO₃, dehydrated in acetonitrile, dried and swelled in digestion buffer (50 mM NH₄HCO₃, 5 mM CaCl₂ and 12.5 ng/μl trypsin Gold (mass spectrometry grade; Promega, Madison, WI, USA). Digestion was performed overnight at 37°C and the peptides were extracted by 1 change of 20 mM NH₄HCO₃ and 3 changes of 5% formic acid in 50% acetonitrile. The samples were finally dried and the peptides resuspended in 6 μl of buffer A (water/acetonitrile/formic acid, 97.7/2/0.3, V/V/V). The peptides were separated using an inert nano LC system composed of a Famos micro autosampler, a Switchos micro column switching module and an Ultimate micro pump from LC Packings (San Francisco, CA) before MS analysis. Of the in-gel digested samples 5 μl was preconcentrated and desalted on a 300 μm inner diameter x 5 mm nano-precolumn (LC Packings) packed with 5 μm C18 PepMap100 material. A 75 μm inner diameter x 15 cm nano-column packed with 3 μm C18 PepMap100 material was used to separate the peptides. Elution from the column was made with a gradient by mixing decreasing volumes of buffer A with increasing volumes of buffer B (water/acetonitrile/formic acid, 9.7/90/0.3, V/V/V). The peptides were eluted into the nano electrospray ion source of the quadrupole time-of-flight (Q-TOF) Premier mass spectrometer (Waters). MS survey scans were acquired using MassLynx 4 SP4 (Waters) from m/z values between 450-1500. The instrument was operated in a data-dependent MS to MS/MS switching mode. Doubly, triply and quadruply charged peptide ions detected in MS survey scans triggered a switch to MS/MS for obtaining peptide fragmentation spectra with an interval of m/z values between 50-1800. [Glu¹]-fibrinopeptide B (GFP), 300 fmol/μl, was used as lock mass injected at a flow rate of 300 nl/min. GFP was also used to calibrate the TOF unit. Raw data were processed using ProteinLynx GlobalServer 2.1 (Waters) with processing parameters: Background Subtract: Normal, Background Threshold: 35%, Background Polynomial: 5, Smoothing Type: Savitzky-Golay, Smoothing Iterations: 2, Smoothing Window: 2 channels, Deisotoping Type: Normal, Deisotoping Threshold: 1%. The processed data were used to search the mouse fraction of the Swiss-Prot database (release 2011_11 or 2013_05) using the on-line version of the Mascot MS/MS Ion Search facility (Matrix Science, Ltd., http://www.matrixscience.com) [10]. Searching was performed with doubly, triply and quadruply charged ions with up to 2 missed cleavages, a peptide tolerance of 20 ppm, one variable modification, Carbamidomethyl-C, and an MS/MS tolerance of 0.05 Da. Spectra of dubious identifications were evaluated manually and omitted if they were of insufficient quality. Contaminating peptides and cross-contaminating peptides from previous samples including keratins, trypsin, BSA and casein were disregarded. At least one ‘bold red’ peptide with scores giving a less than 5% probability that the observed match was a random event was required in the search for protein hits. All peptides for these hits are reported. If the first search in the mouse fraction of the Swiss-Prot database did not give any hit an additional search in the whole database was performed.

Liver

In total 22 spots were found to be differentially expressed in α1,3-galactosyltransferase gene KO mice relative to wt mice as shown in Figure 2 panel A. Eight spots were upregulated, marked with green numbers, and 14 spots where downregulated, illustrated by red numbers. All spots were excised and subjected to mass spectrometry identification as summarised in Table 1. Eighteen spots were successfully identified. Of these, 12 were found to contain one protein that accounts for the observed change in expression. Six spots contained more than one protein (Nos. 0511, 2514, 3113, 6207, 6311 and 7104) in which case, we cannot with certainty conclude which of them accounts for the observed change in expression level. Interestingly, in the vast majority of cases we observed a major deviation of the observed molecular mass by 2D-PAGE with the theoretical molecular mass of the proteins, as illustrated in Figure 3. α-tubulin has a molecular mass around 50 kDa but spot 2514 migrated with a molecular mass below 40 kDa (Figure 3A). A map of the identified peptides showed that spot 2514 contained an N-terminal fragment of tubulin. A change in the concentration of a protein fragment may occur as a result of a change in the synthesis as well as a change in the degradation as it is the case with a full-length protein. Thus, it seems that for some proteins there is a substantial change in protein turnover in the KO liver.

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Figure 3. Spot No. 2514 is a fragment of α-tubulin

A) Spot 2514 migrates with a molecular mass below 40 kDa. The identified peptides from the spot are distributed in the N-terminal of α-tubulin and spot 2514 thus corresponds to a fragment of α-tubulin, since α-tubulin possesses a molecular mass around 50 kDa.

https://doi.org/10.1371/journal.pone.0080600.g003

Identified upregulated protein spots included adenosylhomocysteinase fragment (2001), serum albumin (3812), 2-hydroxyacyl-CoA lyase 1 (5813) and isocitrate dehydrogenase [NADP] cytoplasmic fragment (6414). Downregulated spots included heat shock cognate 70 kDa protein 3 fragment (0413), glutathione S-transferase P 1 (2203), betaine-homocysteine S methyltransferase 1 fragment (3209), aldehyd dehydrogenase fragment (3210), myosin-9 fragment (4206), electron transfer flavoprotein subunit α fragment (6102), arginase-1 fragment (7202) and urocanate hydratase (8803). Only one protein of all identified proteins in Table 1 contained a putative N-glycosylation site, namely carboxylesterase 3 (spot 6207).

Pancreas

In total 39 differentially expressed spots were detected by 2D-PAGE analysis (Figure 2, panel B). All spots were excised and resulted in identification of 17 spots as summarised in Table 2. All of these were found to contain a single identification and about a third of them contained fragments of the proteins. Thus, there is also a substantial change in protein turnover for some proteins in the KO pancreas tissue. Of upregulated proteins, the following eight were identified; albumin fragment (2602), tensin-3 fragment (7702), serotransferrin (7808), chymotrypsin-like elastase family member 1 (8305), NADH dehydrogenase [ubiquinone] flavoprotein 1 (8604), ATP synthase subunit alpha, mitochondrial (8615), aconitate hydratase, mitochondrial (8802) and 3-hydroayacyl-CoA dehydrogenase type 2 (9303). Identified downregulated proteins were 78 kDa-glucose-regulated protein (1811), apolipoprotein A-1 (2315), glutathione S-transferase P1 (3215), 40S ribosomal protein S12 (5005), proteasome subunit β type-3 (5208), serum albumin fragment (5413), myeloperoxidase fragment (5415), GTP-binding protein SAR1a (6202) and serum albumin fragment (6304). Of all identified proteins in Table 2, only four contained putative N-glycosylation sites, i.e., myeloperoxidase, serotransferrin, receptor-type tyrosine-protein phosphatase α and chymotrypsin-like elastase family member 1.

Conclusion

We have analysed two different tissues of α1,3-galactosyltransferase KO mice and revealed that deletion of this gene that directly affects the expression of the α-Gal epitope affects a number of proteins with a wide variety of functions. Thus, it seems that protein turnover, i.e., synthesis and/or degradation, for some proteins is greatly influenced in tissues of the KO mice.