When access to freezing or refrigeration is not possible, desiccation is a simple mechanism to preserve protein samples collected during field expeditions. Toward developing a protein preservation method specifically for vespine trophallactic fluid, which could have applicability to field samples generally, we began with a protein preservation trial in the laboratory. We first generated a complex protein mixture with which to test methodologies that would assess protein preservation for subsequent mass spectrometry analysis. We made a hypotonic extract of HEK 293 cells. Cleared extracts were then aliquoted in 10μl volumes into multiple 0.7ml microcentrifuge tubes, some containing 25 μg or 50μg of dried SDS. We reasoned that SDS could more rapidly desolvate protein constituents and would remain compatible with SDS-PAGE upon return to the laboratory. Aliquots were then either frozen at −80°C directly, or placed into a simple hand-made, portable desiccation chamber (see methods) and left for 3 weeks at a constant temperature of 37°C to mimic the potentially warm temperatures and duration of an extended field expedition. The samples were then resuspended in protein sample loading buffer of different SDS concentrations such that the final SDS concentration for each sample was equal and at 2%. The samples were heated to 95°C for ten minutes and then subjected to SDS-PAGE and the results are shown in . Using the frozen sample as reference, all desiccated samples showed a similar banding pattern when stained with Coomassie blue, although the SDS-containing samples showed a slight reduction in total protein. To determine if proteomic identification would be compromised in the desiccated samples, two regions of the gel were chosen where some bands appeared diminished, particularly in the 50μg SDS lane. Region 1 was chosen for its complexity, higher molecular weight, and also because it was not dominated by a major protein species. Together these attributes would make for a fair challenge to test the efficacy of protein preservation in terms of peptide/protein identification and one might expect some reduction in peptide/protein identification in the SDS-containing samples as lower protein abundance could result in proteins falling below the level of detection of the mass spectrometer. Region 2 was chosen as a less complicated sample and one with largely high abundance proteins. Thus, a small drop in protein abundance would have little effect on protein or peptide identification as the analyte concentrations would be sufficiently above detection levels.
Figure 1 Desiccation methods for protein preservation of a complex cell extract for MS-based identification. (A) SDS-PAGE and analysis of HEK 293 hypotonic whole cell extract preserved by freezing or desiccated in the presence and absence of SDS as indicated. (more ...)
The indicated regions were cut from the gel, cubed and subjected to in-gel tryptic digestion and LC-MS/MS analysis in a linear ion trap-orbitrap hybrid mass spectrometer. Mass spectra were searched against a concatenated forward and reverse human IPI protein database 14
. Top SEQUEST hits were plotted using Xcorr values and the difference in ppm of the observed and theoretical precursor masses (). These parameters as well as the ΔCn scores (Supporting Information Fig. 1
) were used to filter the data to a less than 0.01% false discovery rate 14
. The total number of peptides identified in each region is indicated in and the percentage of peptides mapping back to proteins common across samples for each region is indicated in . These data suggested that there was an apparent small decrease in peptide identification in the SDS-containing samples from Region 1. However, the number of protein identifications across sample conditions was still rather constant. Taken together these desiccation methods were sufficiently strong at protein preservation for proteomic identification so as to warrant a test of our method on a true field sample. Larval trophallactic fluid of the yellowjacket Vespula vulgaris
, a species common in Vermont, was collected as described in the methods. Aliquots were made into empty tubes or tubes containing SDS as was done for the HEK 293 cell extract, except some aliquots were placed directly on dry ice that had been carried into the field. Additionally, other aliquots were placed into tubes containing a dried cocktail of protease inhibitors as another possible preservation method. Aliquots that were not frozen were placed into the portable desiccation chamber, returned to the lab, and the chamber was placed at 37°C for three weeks. Samples were then resuspended in sample buffer and subjected to SDS-PAGE as for . Coomassie staining revealed that each protein preservation method afforded significant protection from proteolysis (). However, some measure of degradation was observed particularly in the lane without additives. Given that larval trophallactic fluid is known to contain proteolytic activity 11, 15, 16
this was not unexpected. The sample preserved with SDS provided the strongest protection because in addition to more rapid desiccation, it was likely inhibitory to enzymatic activity. Although not as strong as the SDS, the protease inhibitors also afforded protection. In a separate experiment we determined that protease inhibitors kept in the desiccation chamber for three weeks at 37°C provided similar protection to freshly dried protease inhibitors (Supporting Information Fig. 2
), but that dried protease inhibitors at room temperature for six months had significantly reduced protection (not shown) consistent with the manufacturer’s product details.
Figure 2 Preservation of field samples for MS-based protein identification. (A) SDS-PAGE analysis of V. vulgaris larval trophallactic fluid following a 3 week preservation period by either freezing or desiccation, with or without SDS or protease inhibitors (P.I.) (more ...)
We next sought to determine if protein identification could be made using the field samples that had been subjected to our preservation regimes. The gel bands indicated in were cut from the gel and subjected to in-gel tryptic digestion. LC-MS/MS analysis of recovered peptides was performed as for the HEK 293 cell proteins. Given that V. vulgaris
is a non-model organism without a sequenced genome, it was not surprising that SEQUEST searches against the Apis mellifera
(honey bee), Drosophila melanogaster
, Nasonia vitripennis
(jewel wasp) and a non-redundant database did not reveal a confident identification. We then first analyzed the data from the most preserved, control sample (, lane 1) using the de novo
sequencing software program PepNovo 4
. PepNovo results were trimmed to remove contaminant sequences such as trypsin and keratins, and peptide sequences achieving the top 200 PepNovo scores were subjected to BLAST searched in batch format against the A. mellifera
or the N. vitripennis
protein databases. The results of the BLAST searches, each giving the top ten best matches, were curated manually. The A. mellifera
or N. vitripennis
protein databases exhibited five proteins that had multiple unique peptide hits. The remaining PepNovo peptides with a score greater than six were then batch BLASTed and examined for the presence of one of the top five proteins identified in the top 200 batch BLASTs. The protein with the greatest number of unique hits had 14, while the four remaining proteins had four or less. This top protein was “larval-specific very high density lipoprotein,” an A. mellifera
protein with a predicted molecular weight of 150 kDa, consistent with the molecular weight of the protein excised from the gel as determined by SDS-PAGE (). The 14 unique peptide hits found for this protein were redundantly found, at least in part, a total of 76 times. We manually inspected each MS/MS spectrum for these 14 unique tryptic peptides to validate the PepNovo assignments and in some cases to extend the PepNovo sequences when PepNovo left an amino- or carboxyl-terminal mass deficiency (gap) off the precursor mass. shows an example MS/MS spectrum for one such peptide where we were able confidently to extend the PepNovo-identified sequence six additional amino acids. Manual validation was greatly aided by having acquired high resolution mass measurements during the MS/MS scans as can be appreciated by examining the theoretical and observed singly- and doubly-charged daughter ions for the MS/MS spectrum shown in (Supporting Information Table 1
). Furthermore, the charge state of each fragment ion was readily determined owing to the high resolution measurements in the orbitrap. Importantly, similar results were obtained when analyzing the mass spectra generated from the protein bands from lanes two and three shown in . Indeed most of the 14 unique tryptic peptides found from the peptides derived from the band in lane one were found amongst the peptides originating from the bands in lanes two and three. The 14 V. vulgaris
tryptic peptides are aligned with homologous sequences in A. mellifera
and shown in . Supporting Information Figure 3
shows the complete amino acid sequence of the larval-specific very high density lipoprotein from A. mellifera
and the location of the identified orthologous tryptic peptides from V. vulgaris
. Supporting Information Figure 4
shows a second manually-validated and annotated MS/MS spectrum with theoretical and measured mass values shown in Supporting Information Table 2
. Of note, despite the high degree of similarity between the V. vulgaris
and A. mellifera
sequences, none of the tryptic peptides was identical between these species, thus precluding the identification of these peptides using a SEQUEST search and the A. mellifera
protein database. Taken together, these results describe methodology capable of preserving field samples for many weeks in warm temperatures. Furthermore, we have shown that using these methods, protein preservation is sufficient for protein identification using LC-MS/MS and subsequent database searching or via de novo
sequencing methods when protein databases are not available.
Having established preservation methods facilitating the identification of proteins from field samples, we next sought to determine if these methods were capable of preserving enzymatic activity so as to measure, at least qualitatively, the digestive capacity of larval and adult vespine trophallactic fluid. We reasoned that our methodologies to preserve enzymatic activity could not employ any of the additives used simply for protein preservation, as SDS or protease inhibitors would either fully inhibit or artificially alter proteolytic activity. We procured purified myosin heavy chain and asked if fresh vespine trophallactic fluid could degrade myosin, as myosin would be a major protein in vespine diets. Larval and worker trophallactic samples were collected from a colony of Vespula germancia located in Vermont. Samples were frozen in the field on dry ice and upon returning to the laboratory were challenged for the ability to digest myosin. Trophollactic fluid from both larvae and workers showed significant digestive capacity (). We next tested for the presence of proteolytic activity in desiccated field samples. Larval and worker trophallactic fluid from Vespa mandarinia was collected in Japan, desiccated, and upon arrival at the US-based laboratory was subjected to the myosin digestion assay and results are shown in . Again, both worker and larval samples showed digestive capacity. Additional samples were collected and desiccated on field expeditions to Singapore, the Southwestern United States, Vermont and Japan. The results are shown in . To date, each species and animal type (workers and larvae) have tested positive for digestive capacity.
Figure 3 SDS-PAGE analysis of myosin proteolytic activity assays performed on samples collected in the field. (A) Trophallactic fluid (T.F.) from V. germanica collected in Vermont, frozen (F) on dry ice, then stored at −80°C prior to myosin activity (more ...)
Summary of the results of myosin proteolytic activity assays from the 13 Vespine species analyzed. Collection sites are in parenthesis.
While the purpose of this report is primarily to describe the methodology by which proteins from field samples can be successfully preserved for identification and enzymatic analysis, our results regarding vespine biology can be variably interpreted. First it is possible that V. orientalis has undergone such evolutionary divergence so as to become unique among vespines in exhibiting an extreme division of digestive labor. If such is the case, it is likely that specific proteolytic enzymes are sufficiently under-expressed in the workers of V. orientalis such that they are reliant on the larvae for digestion and survival. Given the arduous, but not impossible task of de novo peptide sequencing (), a complete proteomic comparison of vespine trophallactic fluids will dramatically benefit by whole genome sequencing of vespine species, a goal we are pursuing. Second, it is possible that all vespine workers can digest proteins, at least to a degree, but some species such as V. orientalis may only be capable of limited digestion and therefore remain reliant on larvae for complete digestion. We are currently attempting to collect samples from V. orientalis workers and larvae to determine the degree to which they digest myosin in our assay. Should V. orientalis exhibit digestive capacity similar to what is observed in other vespines, our qualitative assay will be refined to take on a more quantitative approach. The development of a quantitative assay will be in consultation with phylogenetic relationships drawn from morphological and genomic analyses currently underway in our laboratory on a multitude of vespine species including those listed in . Once these relationships are better understood, we will quantify the digestive capacity of workers and larvae between samples of V. orientalis, our best approximation of its closest living relatives, and more distant living relatives—not hypothesized to exhibit any digestive division of labor—as outgroup controls.