Elucidating the Biochemical Overwintering ... - ACS Publications

ARTICLE pubs.acs.org/jpr

Elucidating the Biochemical Overwintering Adaptations of Larval Cucujus clavipes puniceus, a Nonmodel Organism, via High Throughput Proteomics Martin A. Carrasco,*,† Steven A. Buechler,‡ Randy J. Arnold,§ Todd Sformo,|| Brian M. Barnes,^ and John G. Duman† ‡

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Department of Applied and Computational Mathematics and Statistics, University of Notre Dame, Notre Dame, Indiana 46556, United States § Proteomics Facility, Indiana University, Indianapolis, Indiana, United States University of Alaska, Fairbanks, Alaska, United States ^ Institute of Arctic Biology, University of Alaska, Fairbanks, Alaska, United States † Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana 46556, United States

bS Supporting Information ABSTRACT: Cucujus clavipes puniceus (C.c.p.) is a nonmodel, freeze-avoiding beetle that overwinters as extremely cold-tolerant larvae in the interior boreal forests of Alaska to temperatures as low as 100 C. Using a tandem MS-based approach, we compared the proteomes of winter- and summer-collected C.c.p. to identify proteins that may play functional roles in successful overwintering. Using Gene Ontology (GO) analysis and manual interpretation, we identified 104 proteins in winter and 128 proteins in summer samples. We found evidence to indicate a cytoskeletal rearrangement between seasons, with Winter NDSC possessing unique actin and myosin isoforms while summer larvae up-regulated α actinin, tubulin, and tropomyosin. We also detected a fortification of the cuticle in winter via unique cuticle proteins, specifically larval/pupal rigid cuticle protein 66 precursor and larval cuticle protein A2B. Also, of particular interest in the winter larvae was an up-regulation of proteins related to silencing of genes (bromodomain adjacent to zinc finger domain 2A and antisilencing protein 1), proteins involved with metabolism of amines (2-isopropylmalate synthase and dihydrofolate reductase), and immune system process (lysozyme C precursor), among others. This represents the first high throughput MS/MS analysis of a nonmodel, cold-tolerant organism without a concurrent microarray analysis. KEYWORDS: Cucujus clavipes, freeze avoiding, nonmodel organism, proteomics, high-throughput, beetle, low temperature biology

’ INTRODUCTION The red flat bark beetle, Cucujus clavipes puniceus (C.c.p), is a carnivorous, freeze-avoiding insect that survives extreme conditions during Alaska winters. It overwinters primarily as larvae located under the decaying bark of dead poplar trees, either fallen or standing. The western subspecies is found north of the Brooks Range in arctic Alaska. To avoid freezing, it employs both behavioral and physiological modifications. In fall, insects move to within 30 cm of the base of standing trees to exploit the insulating effects of snow. Physiological changes include extreme dehydration to as low as 28% body water, production of antifreeze proteins (AFPs) and glycolipids, multimolar glycerol concentrations, entry into diapause, and purging the gut to remove ice nucleators.1 3 The dehydration both leads to an increased concentration of AFPs and glycerol and reduces the number of water molecules available to freeze. In midwinter, C.c.p. typically have mean supercooling points (the temperature where freezing occurs) of approximately 40 C; however, r 2011 American Chemical Society

through magnification of several of these physiological adaptations, especially dehydration, some C.c.p. deep supercool and do not freeze at any temperature; instead, their body water vitrifies (turns to glass) at temperatures between 58 and 70 C. Deep supercooling larvae can survive temperatures to at least 100 C.2 Nonmodel organisms, such as C. clavipes, are challenging for use in proteomics because the lack of available transcriptome data makes it difficult to validate changes in protein concentrations with changes in gene expression. However, numerous studies have examined the proteome of nonmodel organisms, primarily through two-dimensional gels.4 11 Shevchenko et al.4 first performed a small scale proteomics experiment on an unsequenced yeast species and successfully identified 15 proteins using de novo sequencing coupled with a homology search, MS-BLAST. Received: May 11, 2011 Published: September 19, 2011 4634

dx.doi.org/10.1021/pr200518y | J. Proteome Res. 2011, 10, 4634–4646

Journal of Proteome Research These authors wrote the MS-BLAST program specifically for MS analysis that has since been used in numerous proteomics studies.5 7,9 13 Samyn et al.7 applied a similar approach with the addition of the FASTS algorithm and identified over 40 novo-proteins in bananas (Musa acuminate) and over 30 proteins in the bacterium Halorhodospira halophila proteome.14 Guercio et al. identified 27 proteins in Bothrops atrox venom and compared these proteins between juveniles, subadults and adults.5 Behr et al. successfully identified 84% of the proteins investigated using a similar approach in Lactobacillus brevis.6 Proteomics of cold tolerance in insects has been investigated using two-dimensional gels.15 Colinet et al.16 found that cold exposure at 4 C in parasitic wasps, Aphidius colemani, resulted in up-regulation of proteins involved in energy metabolism, protein chaperoning, and protein degradation. They also found evidence that cytoskeleton components could play a role in insect survivorship during times of fluctuating cold exposure. Using subtractive two-dimensional gels, Li et al.17 found that diapausing flesh flies, Sarcophaga crassipalpis, possess 37 upregulated proteins that included heat shock proteins. Fortythree proteins were down-regulated or absent in diapause, including phosphoenolpyruvate synthase and fatty acid binding protein. Li and Denlinger18 examined the proteomes of flesh flies that underwent rapid cold hardening. They selected 14 high abundance proteins for identification and found that three increase in abundance, including ATP synthase subunit alpha, a small heat shock protein, and tropomyosin-1 isoforms 33/34. The other 11 proteins either are missing or decreased in abundance including proteins involved in energy metabolism, protein degradation, transcription, actin binding, and cytoskeleton organization. To date, there has been one publication that describes a high throughput proteomics approach on nonmodel organisms. Grossman et al.13 charted the proteomes of spinach, bell pepper, and Cassava plastids using database search software on uninterpreted MS/MS spectra coupled with de novo sequencing and a modified cross species database search of a sequenced organism. Up to 20% more groups of homologous proteins were identified than by standard uninterpreted MS/MS spectra correlation software. To identify proteins that may be critical to winter survival this study applies high throughput proteomics to a nonmodel insect, C.c.p. Proteomes of summer larvae were compared to those of winter larvae that supercool to low temperatures of approximately 40 C, but do not vitrify (i.e., they eventually freeze and do not deep supercool), hereafter referred to as Winter nondeep supercooling (NDSC) larvae. This study revealed proteins unique to, or up-regulated in, Winter NDSC relative to summer and others unique to, or up-regulated in, summer relative to NDSC winter. In this case a proteomics approach is preferable to transcriptomics because C.c.p. produces the various proteins needed for winter survival at varying times throughout the late summer and autumn, and consequently a transcriptomics approach, theoretically, does not provide as complete an accounting of variations in protein content between Winter NDSC and summer. For example, antifreeze proteins are produced by Alaska C.c.p. only during a two week period beginning in mid-August. AFP transcripts are not present later in the autumn or in winter. Here we investigate biochemical adaptations through proteomic analysis that may contribute to C.c.p.’s cold hardiness.

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’ METHODS AND MATERIALS Insect Collection and Microhabitat Characteristics

C. c. puniceus larvae (in stages that ranged from 1 to 2 years) were collected from under the bark of dead Poplar spp. trees near Fairbanks, Alaska. Summer samples were collected in June 2008 and brought directly to the lab for measurements of freezing points or frozen for later proteomics analysis. Larvae to be used for Winter NDSC samples were collected in late September, 2007 and placed in plastic food storage containers (20 cm 15 cm 10 cm; 20 150 insects per container) perforated for gas exchange and provided with moist bark from their host trees.2 Insects were allowed to acclimatize to local outdoor conditions in an undisturbed wooded area on the University of Alaska Fairbanks campus until analyzed. Winter NDSC samples were processed on 12/21/2007, 1/28/2008, 2/5/2008, and 2/6/2008. By November they are fully acclimated, and we began to measure supercooling points (SCPs) to determine those that are deep supercooling (DSC) and nondeep supercooling (NDSC); this manuscript deals only with those that are NDSC. Air and microhabitat temperatures at this location were monitored and recorded using Hobo Pro Series data loggers and downloaded with BoxCar Pro 4 software (Onset Computer Corporation, Bourne, MA). Supercooling Points

Insects were collected from the containers at different time points throughout the winter of 2007 2008 and brought inside the laboratory at the Institute of Arctic Biology in Fairbanks to assess their supercooling points (SCPs). Larvae were tested for individual SCP by wedging a thermocouple junction (copperconstantan, 36 gauge) against their body in a 0.6 mL plastic tube. Thermocouple leads were attached to a computer-controlled multichannel thermocouple thermometer (Iso-Thermex, Columbus Instruments, Columbus, OH) that recorded the temperature every 5 s in 16 channels. Closed tubes were placed inside a covered 500 mL glass beaker that was mostly submerged in an alcohol water cooling bath. Bath temperature was reduced at a rate of 0.2 C min 1 after the insects temperature equilibrated to 0 C. Each thermocouple attachment was visually inspected before and after each run to ensure the thermocouple junction was in direct contact with the insect. The lowest body temperature recorded at the release of the latent heat of fusion, as indicated by an exotherm in the temperature recording, was recorded as the SCP, the temperature at which the insect froze. Only those winter larvae that froze, referred to as nondeep supercooling (Winter NDSC), were used in this proteomics study and compared to summer. Protein Extraction/Preparation

A diagram of how proteins were extracted and prepared for analysis is given in Figure 1. After SCPs were determined, insects were frozen at 80 C and later shipped on dry ice to the University of Notre Dame where protein extraction occurred. Larvae were pooled: Winter NDSC group (N = 38) (total of 280 mg); Summer group (N = 84, mass = 1300 mg). Larvae were placed in a mortar and covered with liquid nitrogen until it sublimed. Lysis buffer (8 M urea, 2% CHAPS, 10 mM DTT) was added to cover the larvae before they were ground to a powder using a pestle. An incubation period of five minutes at room temperature allowed the liberated proteins to solubilize, and then the protein solution was transferred to a 50 mL Eppendorf tube. A clinical centrifugation (Dynac Centrifuge, Clay Adams, 4635

dx.doi.org/10.1021/pr200518y |J. Proteome Res. 2011, 10, 4634–4646

Journal of Proteome Research

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the low molecular weight proteins. Each lane was sectioned vertically to acquire a portion of the entire population of proteins in the lane. The in gel trypsin digest and extraction was carried out using the protocol of Williams and Stone.19 The extracted peptides were lyophilized completely and shipped on ice to the Indiana University Proteomics Facility (IU-Bloomington) for nano LC MS/MS analysis. Each sample was electrophoresed and trypsinized twice and then subjected to tandem nano LC MS/MS analysis for a total of two tandem MS runs per sample. Tandem MS

Figure 1. High throughput proteomics workflow. Larvae from each season, Winter NDSC and Summer, were pooled and the ensuing global protein samples were collected (Winter NDSC N = 38; Summer N = 84). Each sample was clarified on a standard sized, precast SDS-PAGE gel using standard electrophoresis conditions. Each lane was then cut into 1 mm thick strips along the length of the lane, and then these strips were cut into 2 equal sized strips, high and low molecular weight proteins. In-gel trypsin digests were performed, and the peptides were extracted immediately afterward. The peptides were run on a tandem MS machine, CID-IT, with a RPLC gradient in-line and upstream of the analyzer. Ensuing data were matched to Sprot using MASCOT. Also, MASCOT generic format files were created and de novo sequenced using PepNovo and the corresponding decoy sequence databases were constructed (target peptides with the sequences reversed). Contaminant peptides were removed by matching to an empty lane, negative control. Quality, sequenced peptides were matched to Sprot using FASTS and MS-BLAST. Matches from all 3 homology search programs that were above the threshold were entered into a master database. Those proteins that had at least 2 corresponding peptide matches were considered present. The databases were then compared using Gene Ontology (GO) and manual interpretation methods.

Each sample was diluted by adding 20 μL solvent A (97/3/0.1 water/acetonitrile/formic acid) and placed in an autosampler vial. Five microliters of each sample was injected onto a 15 mm 100 μm i.d. trapping column packed in house with 5 μm of 200 Å pore size Magic C18AQ material and flushed with solvent A. Peptides were then separated by reversed-phase nanoflow chromatography on a 150 mm 75 μm i.d. analytical column pulled to a tip and packed in house with 5 μm of 100 Å pore size Magic C18AQ material (Michrom Biosciences, Auburn, CA) using a 120-min gradient from 3 to 40% solvent B (0.1% formic acid in acetonitrile) at 250 nL/minute (Eksigent NanoLC-2D, Dublin, CA). Peptides eluting from the column were electrosprayed directly into the source of an ion-trap mass spectrometer (LCQ Deca XP Plus, ThermoFinnigan, San Jose, CA) where a continuous cycle of one mass spectrum followed by two tandem mass spectra of the two most intense precursors were acquired. Dynamic exclusion was enabled such that each precursor m/z was selected for tandem mass spectrometry no more than twice over a one minute window.

’ SPECTRAL ANALYSIS MASCOT

Tandem mass spectra were extracted from the raw data file and searched against the entire Swiss-Prot database using a licensed copy of Mascot.20 Fixed modification of carbamidomethyl cysteine, variable modifications of protein N-terminal acetylation and methionine oxidation, and one missed trypsin cleavage site were allowed in the search. Peptide identifications with Mascot scores greater than or equal to 35 and proteins with a combined peptide score greater than or equal to 50 were considered valid. De novo Sequencing

Protein samples were prepared for tandem mass spectrometry analysis by standard sized SDS-PAGE gel electrophoresis (72 mm length lanes). 40 μg of each sample was electrophoresed on a 10% Tris-HCL Ready Gel (Bio-Rad, Melville, NY) using the Bio-Rad Mini-Protean II electrophoresis system at 130 V until the dye front reached approximately 1 cm from the edge of the gel, usually after 90 min.

De novo sequencing dates back over 30 years.21 Mascot generic format files (mgf) were generated for each fraction via Pepnovo v3.00 (CSE Bioinformatics Group, University of California at San Diego, CA) with the following parameters: post-translational modification (PTMs) C+57:M+16 and digest, trypsin. PepNovo was used due to its robust nature on ion trap-based MS machines22,23 and prevalence in the literature.9 11,13,24 After sequencing, peptides that were less than six amino acids in length or that had a PepNovo score of e0 were removed. The remaining quality peptides were incorporated into FASTA format files, referred to as the target files. Decoy files were generated by reversing the sequence of peptides. These were incorporated into separate FASTA format files and used to determine the false discovery rate (FDR).

In Gel Trypsin Digest and Extraction

’ HOMOLOGY SEARCHES

Parsippany, NJ) at 4000 rpm clarified the preparation and the supernatant was aliquoted in 1.5 mL microcentrifuge tubes. Protein concentration was assessed using the Bio-Rad Protein Assay kit (Bio-Rad, New York, USA). Electrophoresis

A 1 mm strip was cut vertically down the length of each lane and then this strip was cut horizontally into two equal portions; one portion containing the high molecular weight and the other

Contaminant Filtering

Studies have shown that filtering out contaminants results in less false positive hits and improves the confidence of identification

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Journal of Proteome Research of minor protein components.10,11,13 To exclude contaminants, a negative control was performed. This negative control consisted of an empty lane from the SDS-PAGE gel that was run through our proteomic pipeline and therefore should contain contaminant proteins introduced through our workflow, such as trypsin. The empty lane was cut lengthwise in a 1 mm strip and this was cut into two portions, high and low molecular weight. Each gel slice was in gel trypsin digested and underwent peptide extraction. The resulting sample was run on the tandem MS in the same manner as the experimental samples and a MASCOT search was run. The resulting data packets were de novo sequenced and searched against the Swiss-Prot database using both FASTS34 and MS-BLAST. Positive matches that exceeded the threshold scores, as determined by decoy database analysis, were compiled in a database. Subsequent experimental samples were searched against this contaminant database using the FASTS34 algorithm immediately after de novo sequencing and prior to homology searches. Positive matches to the contaminant database were removed and the resultant data packets devoid of contaminant peptides.

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Table 1. Summary of Proteomics Workflow peptides sample

proteins

total detecteda orphan matchesb matchedc

totald

Summer

8821

294

720

128

Winter NDSC

9807

292

621

104

a

All of the peptides detected by the MS detector. Not all peptides were included in homology searches, including those not sequenced and other peptides that were less than 6 amino acids long. Peptides included in the homology searches were matched either above or below threshold; peptides below threshold were removed. The peptides above threshold are represented in the Orphan matches and Matched columns. b Single peptides above threshold. The corresponding protein only had this single peptide match and did not meet our requirements for inclusion. c Peptides matched above threshold to proteins that had at least 2 corresponding peptides. d Number of proteins identified; related to the “Matched peptides” category.

FASTS

Target and decoy files were searched against the Swiss-Prot database using the FASTS34 software25 (http://fasta.bioch. virginia.edu/fasta_www2/fasta_list2.shtml) using a cross species database search. The FASTS software has been used in previous proteomics studies.7 14 The FDR was determined by calculating the E-value of the decoy search that gave e0.05 FDR.26,27 Matched peptides above the threshold score were removed and the unmatched peptides were searched against MS-BLAST. MS-BLAST

Peptides from both the target and decoy files were searched against the Swiss Protein database using the MS-BLAST software hosted on EMBL (http://dove.embl-heidelberg.de/Blast2/ msblast.html). Peptides were input into the server in sets of 50 with the following parameters; unique peptides, 50; score table, 100; matrix, PAM30MS; filter, none; echofilter, on; Expect, 100; Cutoff, default; strand, both; descriptions, 500; alignments, 500; no histogram; parsed HTML, on; advanced options, -nogap hspmax 100 -sort_by_totalscore -span1. Results were exported as text files and parsed using the R software with the top scored match for each peptide being selected. The FDR was set at e0.05 using the decoy files.26,27

Data Compilation/Gene Ontology Analysis and Manual Interpretation

Peptide matches that satisfied the threshold score in each analysis were compiled into a master database for Winter NDSC and another for Summer. Proteins that had a minimum of two peptides matched were retained and those with less than two were discarded. Each of these peptides was required to consist of at least six amino acids. The accession numbers for the peptides that matched these criteria were compiled in a text file, one for Winter NDSC and another for Summer, and categorized using the Gene Ontology database, gene_association. goa_uniprot, on a Unix server. The resulting categorizations were used to make comparisons between the Winter NDSC and Summer samples through the Web Gene Ontology interface (http://wego.genomics.org.cn/cgi-bin/ wego/index.pl). An online Pearson Chi Square test was used to assess significant differences between the two samples (P e 0.05).

Figure 2. Gene Ontology venn diagram for Summer and Winter NDSC samples, including all matches from Biological Process, Cellular Component, and Molecular Function. The number next to the sample name is the total number of GO categories in that sample.

Quantification of proteins was performed using spectral counts,28 32 and differences in expression were determined by PepC software33 using default parameters. Manual interpretation of the results was performed in two manners, on abundant proteins/peptides and on the entire protein set for each sample. Abundant proteins, defined as proteins possessing eight or more peptides in at least one sample, were placed in a separate database for manual interpretation. Although a protein with abundant peptides may result from a high concentration of the intact protein, other factors contributing to abundant peptides may include the ionizability of the peptide and the number of trypsin cleavage sites in the protein. Variants of proteins and spectral abundance (using PepC) were examined. For the manual interpretation on the entire data sets, accession numbers were examined between both databases using a query in Microsoft Access. Matches in both databases were filtered out while unmatched accession numbers in each sample were recorded in separate tables.

’ RESULTS General Results

Winter NDSC larvae collected in Fairbanks, AK showed a marked decrease in mean SCP relative to their Summer counterparts, 35.5 C and 5.6 C, respectively. The general results from our proteomics workflow are given in Table 1. Nearly 1000 more peptides were present in the Winter 4637



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Figure 3. Graphical representation of GO categories between Summer and Winter NDSC (GO level 2 shown). (A) Biological Process. (B) Cellular Component. (C) Molecular Function. * Represents significant difference (p e 0.05) in expression between Summer and Winter NDSC as determined by a Pearson Chi Square test. The number beside the * represents the number of GO level 5 categories that are differentially expressed between Summer and Winter NDSC.

NDSC than the Summer sample (complete lists of sequenced peptides available as Supplemental Table 1, Supporting Information), but more peptides were matched to proteins in the Swiss-Prot database in the Summer, almost 100 more. This resulted in 24 more proteins identified in the Summer than the Winter NDSC. Both samples had approximately equivalent numbers of orphan matches (these are peptides matched to a protein above threshold but without another peptide to support it).

Gene Ontology Comparisons

To make equivalent analyses between samples, we employed the controlled vocabulary of protein classification afforded by the Gene Ontology (GO). Proteins that met our criteria in each sample, Summer and Winter NDSC, were placed in separate databases and classified through GO. The resulting classifications were compared between samples and illustrated in Venn diagrams (Figure 2). When redundant GO categories were removed, thus 4638



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Table 2. Differential GO Expression between Summer and Winter NDSC Cucucjus clavipes puniceus GO level 5 classb

summer countc

Winter NDSC countc

E-valuea

GO level 2 classb

Cellular Component GO:0005576 extracellular region

24

10

0.026

Extracellular region

GO:0016459 myosin complex

9

18

0.049

Macromolecular complex

GO:0016460 myosin II complex

9

18

0.049


GO:0032982 myosin filament

9

18

0.049


Molecular Function GO:0022804 active transmembrane transporter activity

24

11

0.043

Transporter Activity

GO:0016788 hydrolase activity, acting on ester bonds

13

3

0.018

Catalytic activity

GO:0015075 ion transmembrane transporter activity GO:0004518 nuclease activity

26 12

12 3

0.037 0.028

Transporter Activity Catalytic activity

GO:0022892 substrate-specific transporter activity

31

16

0.047


GO:0022857 transmembrane transporter activity

27

13

0.043


Biological Process GO:0006066 alcohol metabolic process

10

2

0.028

Metabolic process

GO:0009309 amine biosynthetic process

3

10

0.036

Metabolic process Metabolic process

GO:0009308 amine metabolic process

6

14

0.047

13

3

0.018

Cellular Process

GO:0044106 cellular amine metabolic process GO:0006519 cellular amino acid and derivative metabolic process

3 4

14 14

0.004 0.011

Metabolic process Metabolic process

GO:0006520 cellular amino acid metabolic process

3

14

0.004

Metabolic process

GO:0032989 cellular component morphogenesis

9

2

0.046

Cellular Process

19

7

0.028

Developmental Proc

7

16

0.036

Metabolic process

GO:0048468 cell development

GO:0048869 cellular developmental process GO:0042180 cellular ketone metabolic process GO:0044265 cellular macromolecule catabolic process

12

3

0.028

Metabolic process

GO:0006936 muscle contraction

10

20

0.038

Multicell Org Proc

GO:0007517 muscle organ development GO:0003012 muscle system process

9 10

2 20

0.046 0.038

Developmental Proc Multicell Org Proc

GO:0016053 organic acid biosynthetic process

3

12

0.012

Metabolic process

GO:0006082 organic acid metabolic process

7

16

0.036

Metabolic process

GO:0043436 oxoacid metabolic process

7

16

0.036

Metabolic process

a Significantly different (p e 0.05) categories are shown as deduced from a Pearson chi square analysis. b GO level 5 categories are shown from Biological Process, Cellular Component and Molecular Function as well as the corresponding level 2 categories from Figure 3. c Counts refer to the number of proteins represented in the indicated level 5 category.

leaving each GO category with a lone representative, the samples share roughly half of their GO categories. The Venn diagram illustrates two types of GO categories; shared, 293, and unique, 280 for the Winter NDSC and 283 for the Summer. The shared category may be further divided into two subcategories; differentially expressed between samples and equivalently expressed. Differential Expression (GO Categories)

To assess differential expression, a Pearson chi square test was performed between Summer and Winter NDSC GO categories at level 5 for Biological Process (Figure 3A), Cellular Component (Figure 3B), and Molecular Function (Figure 3C). Level 2 categories are depicted in Figure 3 due to space constraints of depicting GO level 5 results. Significant differences (p e 0.05) between the samples are represented with asterisks. There were a total of 47 differentially expressed categories in Biological Process, six in Cellular Component, and seven in Molecular Function. In Biological Process, the subcategories of developmental process, multicellular organismal process, cellular process and metabolic process were differentially expressed. Cellular

Component had differences in the extracellular region and macromolecular complex subcategories. Finally, Molecular Function had differences in transporter activity and catalytic activity subcategories. Some GO categories that were differentially expressed appeared several times. After factoring out this redundancy, there were markedly fewer categories that were significantly different. Table 2 shows the level 5 GO categories that were differentially expressed and gives the corresponding level 2 GO category. There were a total of 27 differentially expressed, nonredundant GO categories. Winter NDSC had 14 up-regulated categories compared to 13 for Summer, where up-regulated means the level 5 category had significantly more proteins in one sample than the other and does not necessarily relate to the same protein. Seventy-five percent of differentially expressed Cellular Component subcategories (3 of 4) were upregulated in Winter NDSC, 100% of differentially expressed Molecular Function subcategories (6 of 6) were up-regulated in Summer, and 65% (11 of 17) of differentially expressed Biological Process subcategories (8 of 14) were up-regulated in Winter NDSC. 4639



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Table 3. Summer- and Winter NDSC-Specific Proteins Ascertained from GO Category Differential Expression Analysis protein

Summer spectral count

GO level 5 class

Biological Process 2-oxoglutarate dehydrogenase

2

Alcohol metabolic process

14 3 3-like protein

4

Cell Dev Proc

Acyl transferase

2

Organ Acid biosyn proc

Aldehyde dehydrogenase, mitochondrial precursor Galactose-6-phosphate isomerase

6 2

Alcohol metabolic process Cell macromol catab proc

Myoblast determination protein 1

2

Cell Dev Proc

Seryl-tRNA synthetase

4

Amine metabolic process

Shikimate kinase

2


Titin

3

Cell Dev Proc

UDP-N-acetylglucosamine-N-acetylmuramyl-

2

Alcohol metabolic process

2

Cell Dev Proc

(pentapeptide) pyrophosphoryl-undecaprenol N-acetylglucosamine transferase 2 UDP-N-acetylmuramoyl-L-alanyl-D-glutamate2,6-diaminopimelate ligase Cellular Component 14 3 3-like protein

4

CC Extracell Reg

Beta-fructofuranosidase, insoluble isoenzyme 3

2

CC Extracell Reg

Molecular Function Cytochrome c oxidase

3

Transmembrane trans activity

Putative ammonium transporter 1

2

Transmembrane trans activity

V-type proton ATPase

2

Nuclease Activity

Winter NDSC protein

spectral count

GO level 5 class

Biological Process 2-isopropylmalate synthase

2


3-isopropylmalate dehydratase large subunit 2

2


Actin 2

18

Cell Dev Proc

Acyl carrier protein Anthranilate synthase component II

4 2

Organ Acid biosyn proc Amine metabolic process

Dihydrofolate reductase

2


Gamma-glutamyl hydrolase precursor

2


Imidazoleglycerol-phosphate dehydratase

2


Threonine synthase

2


Tagatose 1,6-diphosphate aldolase 1

3

Cell macromol catab proc

Cellular Component Gamma-glutamyl hydrolase precursor Hyaluronoglucosaminidase precursor Lysozyme C precursor Myosin heavy chain, fast skeletal muscle

2 3

CC Extracell Reg CC Extracell Reg

10

CC Extracell Reg

2

CC Myosin Complex

Molecular Function ADP/ATP translocase 2

2

Transmembrane transporter activity

Acyl carrier protein

4

Substrate specific transporter

Interestingly, the extracellular region category was up-regulated in Summer; antifreeze proteins are generally found in this region in vivo and have been previously shown to be up-regulated in winter by a greater antifreeze protein activity (thermal hysteresis value) and a lower mean supercooling point1 (temperature of spontaneous ice nucleation). To our knowledge, antifreeze

proteins are not listed in the Gene Ontology (however, AFP in GO refers to alpha-fetoprotein and/or antifungal protein). Such an omission precludes the analysis of AFPs via a GO approach. Transporter activity was up-regulated in Summer and present in the Winter NDSC. Muscle contraction, muscle system process, and myosin complex were all up-regulated in the Winter NDSC. 4640



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Table 4. Sample Specific GO Categories (Level 5) of Interest Winter class

description

Summer NDSC count count

GO:0009100

Biological Process glycoprotein metabolic process

3

0

GO:0016458

gene silencing

0

4

GO:0006342

chromatin silencing

0

4

GO:0000183

chromatin silencing at rDNA

0

3

GO:0007623

circadian rhythm

3

0

GO:0060004 GO:0002376

reflex immune system process

3 0

0 2

GO:0048511

rhythmic process

4

0

GO:0005677

chromatin silencing complex

0

3

GO:0005200

structural constituent of cytoskeleton

0

4

GO:0042393

histone binding

0

5

Cellular Component

Molecular Function

Development and developmental process were up-regulated in the Summer. Using this approach, we were able to acquire the list of proteins in each GO category that was differentially expressed. Summer had 18 unique proteins (Table 3) whereas Winter NDSC had 14 (Table 3). Proteins unique to Winter NDSC may confer a cryoprotective, or other winter survival, advantage to larvae while the Summer specific proteins may serve as detractors of winter survival, or may simply not be needed in the nonactive, diapausing larvae. The Winter NDSC sample had seven unique proteins that take part in amine metabolic process and three of these proteins were located in the extracellular region. The Summer sample had four unique proteins that take part in the cellular developmental process, three in the alcohol metabolic process, two in the amine metabolic process, and two more in the transmembrane transporter activity class. Sample-Specific GO Categories

In addition to assessing differential expression, we were able to make comparisons between Summer and Winter NDSC based on the presence or absence of GO categories. We manually scanned the GO reports for categories that were present in either Summer or Winter NDSC and were absent in the opposite sample. There were 67 total sample specific categories (Supplemental Table 2, Supporting Information); Biological Process had 44, Cellular Component had seven, and Molecular Function had 16. Of these 67 total sample specific categories, Winter NDSC had 18 while Summer had 49. Both samples had eight sample specific categories under the Molecular Function heading. The Summer sample had five sample specific categories in the Cellular Component heading compared to two in Winter NDSC. The largest discrepancy between samples came under the Biological Process heading; Summer had 36 sample specific categories whereas Winter NDSC had eight. A list of 11 sample specific, unique GO categories of potential interest to cold tolerance is shown in Table 4. Previous research has shown transcription is down-regulated in overwintering insects,34 and in our study there were proteins from four GO

categories pertaining to down-regulation of transcription found only in the Winter NDSC; chromatin silencing complex, gene silencing, chromatin silencing, and chromatin silencing at rDNA. Another category found only in the Winter NDSC, histone binding, may contribute to transcription down-regulation. There is evidence to support cytoskeletal rearrangement during times of cold stress18,35,36 and our Winter NDSC sample exhibited a unique category, structural constituent of cytoskeleton (Table 4). In addition, the Winter NDSC had another unique category; immune system process. The Summer had unique categories pertaining to daily rhythms, such as rhythmic process, circadian rhythm, and reflex. Also, glycoprotein metabolic process was unique to Summer. Manual Data Interpretation

GO represents a method to compare samples using a controlled vocabulary, which facilitates comparison. However, GO has shortcomings, most notably bias toward certain proteins that are more studied than others; these proteins are better annotated and have numerous entries in GO. One well studied protein may have multiple GO categories across the three broad categories while another protein has a single category. To compound matters, another protein may not have a single category in GO, even in model organisms. Since we studied a nonmodel organism, proteins of interest to us may not be annotated in GO. To circumvent this bias, we also interpreted our data manually. We looked first at the abundant proteins/peptides and then we examined sample-specific proteins. Abundant Proteins/Peptides

Abundant proteins were defined as proteins that possessed eight or more corresponding peptides. A protein with abundant peptides may be abundant in Cucucus, though we recognize the possibility that abundant peptides may be prevalent due to greater ionizability of the peptides relative to less abundant peptides or a greater number of trypsin cleavage sites in the intact protein. By examining the most abundant proteins/ peptides, we attempted to find differences in expression levels. The more peptides present for a particular protein, the greater the probability the protein is present; therefore, this analysis allows for a high confidence comparison. Twelve proteins met this criterion (Table 5), with actin and myosin possessing the most peptides. Other proteins that constitute the cytoskeleton are represented as well as proteins involved with translation, structure, phosphorylation, immune function, muscle contraction, transport, and ATP synthesis. Most proteins of abundance in our study possessed various isoforms and/or chains. For example, actin has 13 variants in the Winter NDSC and Summer sample (Table 5). We clustered isoforms and chains of a particular protein together to form a supergroup. In the case of actin, actin was the supergroup while the 13 actin isoforms were the subgroups. Five forms of actin were sample specific to Winter NDSC while Summer also had five (Table 5). Actin was significantly up-regulated in the Winter NDSC. The supergroup alpha actinin was significantly upregulated in the Summer as was the subgroup alpha actinin sarcomeric, though there were no sample specific forms and each sample had three variants (Table 5). Tubulin was present in detectable quantities in the Summer but not in the Winter NDSC (Table 5) resulting in a significant up-regulation in the Summer. There were three variants of tubulin present with a total of eight peptides. Winter NDSC possessed three variants of cuticle protein, all of which were sample specific (Table 5) while Summer possessed one variant of cuticle protein. 4641



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Table 5. Abundant Protein/Peptide Analysis (Sum of Supergroupsa) and Abundant Protein/Peptide Analysis (Subgroupsb) Summer protein

Table 5. Continued Summer spectral

Winter NDSC

count

spectral count

protein Myosin heavy chain, fast skeletal

Winter NDSC

0

2

113

97

0

2

6

0

9 10

17 9

muscled

proteins

spectral count

proteins

spectral count

13

97

13

140

α Actininc

3

46

3

16

Arginine Kinase

1

11

1

5

Myosin heavy chain, skeletal muscle,

ATP Synthase

2

15

3

9

fetald

Cuticle Protein Lysozyme C

1 2

2 7

3 3

9 19

Paramyosin Paramyosin, long form

Mite Allergen

2

10

1

10

Paramyosin, short formd

Myosin

4

127

6

119

Paramyosin

2

19

3

35

Tropomyosin 1d

3

0

Ribosomal Protein

5

13

2

4

Tropomyosin 2d

15

0

Tropomyosinc

3

30

1

3

Tubulinc,d

2

8

0

0

Actin

Summer spectral

Winter NDSC

count

spectral count

protein

Myosin heavy chain, muscle Myosin heavy chain, skeletal muscle, adult 2d

Tropomyosin

0

9

12

3

Phosphorylation/ATP synthesis/translation Arginine kinase

11

5

ATP synthase epsilon chaind ATP synthase subunit beta

0 13

4 3

2

2

ATP Cytoskeleton/structural

synthase

subunit

beta,

mitochondrial precursor

Actinc

14

38

30S ribosomal protein S3Pd

3

0

Actin 1

14

16

40S ribosomal protein S26d

3

0

Actin 2

8

18

Actin 3d

50S ribosomal protein L9d

0

2

8

0

Actin 3-sub 1d


3

0

3

0

Actin 5 Actin 17d

2 3

10 0

50S ribosomal protein L21d 50S ribosomal protein L25d

2 2

0 0

Actin 18d


0

2

3

0

Actin 85C

24

18

Actin 87E

4 d

Actin, alpha cardiac muscle 1

0

Immune related proteins

11

Lysozyme C

5

4

Lysozyme C I

2

4 5

0

10

Actin, clone 211

2

0

Lysozyme C precursord

Actin, cytoplasmic

3

2

Mite allergen Der p 3 precursor

8

10

Mite allergen Der f 3 precursord

2

0

d

Actin, cytoplasmic 1 Actin, cytoskeletal 3d

9 0

5 4

Actin, cytoskeletal 3Bd

0

5

Actin, muscled

0

4

Actin, muscletyped

0

5

32

11

7

2

Alpha-actinin, sarcomericc Alpha-actinin 1 Alpha-actinin 2

7

3

Tubulin beta chaind Tubulin beta 1 chaind

4 2

0 0

Tubulin gamma 2 chaind

2

0

Cuticle protein 32d

0

2

Larval/pupal rigid cuticle protein 66

0

2

Larval cuticle protein A2Bd

0

5

Flexible cuticle protein 12 precursord

2

0

precursord

Muscle contraction/transport Myosin 1

6

Myosin 4d

0

6 2

Myosin heavy chaind

0

10

Myosin heavy chain Ad

2

0

a

Supergroups refer to a cluster of isoforms and/or chains of a protein. b Subgroups refer to isoforms and/or chains of a protein. c Significant differences as analyzed by PepC (P < 0.05; G > 5). d Sample-specific protein.

Myosin was highly abundant in both samples in similar quantities (Table 5). The Winter NDSC sample had six variants of myosin, four of which were sample specific, while Summer had four variants, two of which were sample specific. Winter NDSC possessed three variants of paramyosin, one of which was sample specific (Table 5). The supergroup tropomyosin was significantly up-regulated in Summer. It possessed three variants, two of which were sample specific (Table 5). Winter NDSC possessed three variants of ATP synthase while Summer had two (Table 5). One of these was sample specific to Winter NDSC. Summer had five variants of ribosomal proteins whereas Winter NDSC had two (Table 5). There were no shared variants between samples; however, they did share various subunits of 50S ribosomal protein. Mite allergen protein is expressed equally in both samples, with two variants in Summer and one in Winter NDSC (Table 5). Summer has one sample specific variant of mite allergen. Also, lysozyme C is more abundant in the Winter NDSC sample, which 4642


Journal of Proteome Research has three variants compared to two in the Summer (Table 5). Winter NDSC has one sample specific variant. Sample Specific Proteins

Identified proteins in both samples were placed into databases which were aligned to search for sample specific proteins. There were 85 sample specific proteins in Summer (Supplemental Table 3A, Supporting Information) and 52 in Winter NDSC (Supplemental Table 3B, Supporting Information). Thirteen of these were flagged as potentially interesting for overwintering (Table 4) due to the confidence in multiple matched peptides and their reported functions. Ten are Winter NDSC specific, two are Summer specific, and one is more abundant in Summer.

’ DISCUSSION To prevent freezing in winter, C.c.p. has been shown previously to undergo extensive dehydration, produce beetle type antifreeze proteins and antifreeze glycolipids, concentrate glycerol, enter diapause and purge the gut.1 3 These mechanisms, in part, lower the supercooling points (temperature of spontaneous nucleation, and death) of the larvae to a mean value near 40 C in winter. Further exaggeration of some of these adaptations (dehydration, glycerol concentrations and AFP activity), in response to especially cold temperatures, allows some larvae to preclude freezing at any subzero temperature and to vitrify, that is, to enter a glasslike transition state and not freeze when ambient temperature is lowered to 58 C (the lowest temperature at which we have seen an exotherm, indicating freezing, in this species) or lower. This is referred to as deep supercooling. Though these physiological overwintering mechanisms have been elucidated, there has not been an attempt to characterize the broader biochemical aspect of C.c.p. overwintering. This study provides this more general context and points to some potentially fruitful areas for future research. Several of our protein candidates that are up-regulated in Winter NDSC have previously been identified as participating in overwintering success in other species. In our current work, there are several occurrences when there was convergence toward a protein or group of proteins between the GO and manual analyses (Table 6). Myosin is the pre-eminent example; in previous research on the 13 lined ground squirrel, Spermophilus tridecemlineatus, myosin was shown to be up-regulated during hibernation and cold stress.27 Specifically, ventricular myosin light chain 3 was up-regulated in the heart tissue of the ground squirrels, though the authors did not speculate on the physiological function in relation to overwintering. To our knowledge, myosin has not been shown to vary during cold exposure in insects. Our GO results indicated numerous categories pertaining to myosin that were significantly up-regulated in Winter NDSC larvae, including myosin complex, myosin II complex and myosin filament, though the proteins comprising these three groups were identical. Our manual analysis did not indicate a difference between the supergroup myosin, which consisted of all myosin variants. However, when the variants were considered, Winter NDSC larvae possessed four variants of myosin absent in the Summer, including myosin 4, myosin heavy chain, myosin heavy chain skeletal muscle adult 2, and myosin heavy chain fast skeletal muscle. The tropomyosin supergroup was up-regulated in Summer, with two variants absent in the Winter NDSC; tropomyosin 1 and tropomyosin 2. Tropomyosin regulates actin mechanics and

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is important for control of muscle contraction. In resting muscle cells tropomyosin overlays the myosin binding site on actin. Thus, tropomyosin is considered a cytoskeletal protein, and in contrast to our results, it has been shown to increase during cold stress. Specifically, tropomyosin 1 has previously been shown to increase in brain tissue during rapid cold hardening in the fly Sarcophaga crassipalpis.18 Tropomyosin 2 has not been previously shown to vary due to cold stress. Tropomyosin was down-regulated in Winter NDSC C.c.p. larvae in our current investigation. Again, there may be differences in physiological responses to rapid cold hardening compared to overwintering, which comes about more gradually and involves exposure to lower temperatures. Summer C.c.p. may have more tropomyosin due to their increased activity and need for regulatory proteins for muscle contraction relative to diapausing Winter NDSC larvae. Such down-regulation of nonessential proteins frees up cellular machinery and energy better suited for other vital overwintering proteins. The collective paramyosin supergroup did not differ significantly between samples, but paramyosin short form variant was only present in the Winter NDSC larvae. Paramyosin is a major component of thick filaments in invertebrate muscles and has not previously been shown to vary due to cold stress in any species. Paramyosin may increase in Winter NDSC larvae to fortify the cytoskeleton. The cytoskeleton elements, including tropomyosin, cuticle protein, profilin, microtubules (tubulin), intermediate filaments, and microfilaments (actin, actinin) have been shown to increase during winter in insects in previous studies. Insects with upregulation of cytoskeletal elements include Culex pipiens,36 Aphidius colemani,16 and Sarcophaga crassipalpis.18 C. pipiens up-regulates actin during cold exposure and diapause.36 Kim et al. used Northern blots to show actin genes 1 and 2 were upregulated during diapause and cold stress in C. pipiens.35 In our study, general actin was up-regulated in the Winter NDSC (Table 5). There were five variants of actin that were specific to Summer and an additional five specific to Winter NDSC, including actin alpha cardiac muscle 1, actin cytoskeletal 3, actin cytoskeleton 3B, actin muscle and actin muscle-type. Actinin, which attaches actin to the Z-line in skeletal muscles, varied between samples. The α actinin supergroup was up-regulated in Summer, as was the α actinin sarcomeric protein. Tubulin proteins, which comprise the microtubules, have not been shown to vary in insects or any other animals in response to cold exposure. However, they have been shown to vary in plants. Arabidopsis thaliana up-regulates α tubulin transcript while down-regulating β tubulin transcripts.37 In our study, we detected β and γ tubulin, both of which were only present in the Summer larvae. Cuticle protein has been shown to increase in A. colemani during cold stress.16 Another study showed three types of cuticle proteins increase in the Colorado potato beetle, Leptinotarsa decemlineata, in response to environmental stresses,38 while two of these increase in response to desiccation stress. The presence of cuticle protein in Winter NDSC larvae may play two roles; preventing water loss in dry, winter conditions and preventing cross-cuticular ice nucleation by fortifying the cuticle. Though C.c.p. undergoes extensive dehydration to promote deep supercooling in winter, the mechanism behind it is not well-defined. Cuticle protein may prevent water loss to preserve the minimal amount of water required during diapause. It is not known if cuticle proteins are involved in prevention of cross-cuticular 4643



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Table 6. Manual Interpretation of Proteins of Interest spectral protein

count

specificity of sample

protein

Granzyme C

2

Summer

Unique

Ribonuclease

6

Summer

Unique

Bromodomain adjacent

5

Winter NDSC

Unique

Cytochrome b

6

Winter NDSC

Unique

Dihydrofolate reductase D-lactate dehydrogenase

2 2

Winter NDSC Winter NDSC

Unique Unique

to zinc finger domain 2A

DNA Repair protein

2

Winter NDSC

Unique

Hyaluronoglucosaminidase

3

Winter NDSC

Unique

4

Winter NDSC

Unique

Organic solvent tolerance

3

Winter NDSC

Unique

protein precursor Orotate

3

Winter NDSC

Unique

Ribosome-binding factor A

3

Winter NDSC

Unique

Transaldolase

2

Winter NDSC

Shared

Transaldolase

8

Summer

Shared

precursor Nuclear polyadenylated RNA-binding protein 3

phosphoribosyltransferase

inoculative freezing, but preventing inoculative freezing initiated by external ice across the cuticle is critical to C.c.p. as it is a freezeavoiding species and any ice formation within the larvae is lethal. Though the collective cuticle proteins were not significantly upregulated in Winter NDSC, there were two variants specific to Winter NDSC: larval/pupal rigid cuticle protein 66 precursor and larval cuticle protein A2B. Membrane proteins increase during cold stress in some insects. Qin et al. investigated the transcriptional changes in Drosophila melanogaster due to rapid cold hardening34 and found that 12 membrane proteins increased as a result of cold hardening, while one decreased. Six of the 12 proteins that increased were putatively assigned identities and the other 6 were unassigned. In our work, we were able to investigate the transmembrane transporter proteins group, which decreased in the Winter NDSC larvae. This is in contrast to the increase seen in D. melanogaster during rapid cold hardening and may be due to the difficulties inherent to solubilizing and ionizing membrane proteins. However, D. melanogaster cannot overwinter in cold regions; therefore, membrane proteins may increase in times of rapid cold hardening in insects, including C.c.p., but may decrease during overwintering. C.c.p. enters diapause to overwinter, which greatly reduces the metabolic needs of the organism; therefore, membrane transport proteins may not be as necessary as during times of activity, such as summer. The one membrane protein that was down-regulated in D. melanogaster, deadhead, participates in electron transport. In our work, cytochrome c oxidase is present in Summer larvae and absent in Winter NDSC, though Cytochrome b is present in Winter NDSC and absent in Summer. Cytochromes, of course, participate in cellular respiration. Cytochrome c is more soluble than the other cytochrome variants and is believed to play a role in apoptosis and, consequently, is in development.39 Our GO analysis also indicates that

cellular developmental processes were up-regulated in the Summer larvae. However, cytochrome c has been shown to increase in Culex pipiens during diapause and cold stress through a microarray approach,36 albeit in early diapause. Concerning other transmembrane transporter proteins, a putative ammonium transporter 1 is present in Summer and absent in Winter NDSC. Oono et al. showed a putative ammonium transporter to be up-regulated in Arabidopsis thaliana during times of cold acclimation, though they did not speculate on the physiological reason.37 The membrane proteins specific to Winter NDSC larvae are ADP/ATP translocase 2, previously shown to be up-regulated in Rana sylvatica during freeze stress40 and acyl carrier protein, which is involved in fatty acid synthesis. Cai at al. speculated that ADP/ATP translocase up-regulation during freezing might be associated with cellular energetics. Metabolic proteins have been shown to increase during times of cold stress and diapause in Aphidius colemani16 and C. pipiens,36 yet decrease in the brain of S. crassipalpis.17 In our study, more metabolic proteins were up-regulated than down-regulated in Winter NDSC larvae. Proteins that increased in A. colemani and/ or C. pipiens include arginine kinase and cytochrome c oxidase, both of which were detected in our experiment. However, there was no difference in arginine kinase expression levels between Summer and Winter NDSC larvae. Cytochrome c oxidase was present in the Summer and absent in the Winter NDSC larvae. ATP synthase (subunit α) has been shown to increase during times of cold stress in S. crassipalpis18 and may play a role in regulating cellular energetics during cold stress. In contrast, numerous ATP synthase subunits have been shown to increase in Arctic ground squirrels, Urocitellus parryii, in early arousal from hibernation when compared to late torpor using proteomics.27 However, there were no significant differences between mRNA levels of ATP synthase. In our current study, although there was no significant difference between the collective ATP synthase supergroup, ATP synthase epsilon chain was specific to the Winter NDSC larvae. ATP synthase epsilon is part of the F1 complex, which serves as the catalytic core of ATP synthase. A knockout study of ATP synthase epsilon chain in HEK293 cells resulted in down-regulation of activity and protein levels of the mitochondrial ATP synthase complex, including a down-regulation of F1 subunits α and β.41 ATP synthase epsilon chain was proposed to play an important role in F1 assembly. While our GO analysis indicated that metabolic proteins increase significantly in the Summer larvae, there were several GO subcategories that were up-regulated in the Winter NDSC larvae, including amine biosynthesis/metabolism, amino acid metabolic process, and organic acid metabolism. During periods of dormancy, insects may retain the ability to reshuffle amino acids from peptides or proteins to meet minimal metabolic demands. Amine metabolic process proteins found only in Winter NDSC larvae include 3-isopropylmalate dehydratase and gamma-glutamyl hydrolase precursor, both previously reported to be down-regulated in cold exposure in A. thaliana.37 Alcohol metabolic process, glycoprotein metabolic process and cellular macromolecule catabolic process are up-regulated in the Summer larvae. Alcohol metabolic process proteins included 2-oxoglutarate dehydrogenase, aldehyde dehydrogenase and UDPN-acetylglucosamine-N-acetylmuramyl-(pentapeptide) pyrophosphoryl-undecaprenol N-acetylglucosamine transferase 2. Proteins related to gene expression have been shown to increase in C. pipiens36 during diapause and A. colemani16 during cold stress at 4 C and to decrease in D. melanogaster in response 4644


Journal of Proteome Research to rapid cold hardening,34 though there were some expression proteins that increased in D. melanogaster. Our results indicate that gene expression inhibition proteins increase during cold stress in C.c.p. (Table 4). Gene silencing, chromatin silencing, chromatin silencing at rDNA, and chromatin silencing complex products were unique to Winter NDSC larvae. Our manual data interpretation also gave evidence to support this; bromodomain adjacent to zinc finger domain 2A was found only in the Winter NDSC. This protein binds to nucleic acid to silence transcription. The immune system process GO category was specific to Winter NDSC and was comprised of two proteins, T-cell surface glycoprotein CD1e precursor and B-cell receptor-associated protein 31. Furthermore, our manual data analysis also showed that an immune related protein, lysozyme C precursor, not detected in GO analysis was found only in the Winter NDSC larvae. Lysozymes are glycoside hydrolases that damage bacterial cell walls by catalyzing hydrolysis of membrane bound sugars. C. c.p. resides under the bark of dead and decaying poplar trees and generally prefers moist environments. During the long winter diapause, especially with decreased metabolism and the potential for cuticular damage, there may be an increased possibility of microbial infection and therefore a need for up-regulation of the immune response.

’ CONCLUSION Our workflow allowed us to quantify peptides using a label free method and to make statistical comparisons between samples using readily available software. The most abundant proteins/ peptides were detected in the greatest abundance, though we were able to detect less abundant proteins/peptides. Furthermore, we applied the controlled vocabulary of the GO to a proteomics experiment and made quantitative comparisons between samples. Without a sequenced and annotated genome, we are unable to identify potential species specific proteins and therefore cannot identify novel proteins that may be related to cold adaptation including vitrification. C.c.p. may possess novel proteins or variants of common proteins that contribute to its overwintering success. The novel proteins may have diverged so much from the ancestor protein that the two are almost impossible to link. Furthermore, C.c.p. may have evolved novel variants of common proteins. These variants may be similar enough to the ancestor protein to allow matching but there may be an important change to the active site or other functionally relevant areas. Though the protein identity may be elucidated, the variation will not be identified using our approach. Genomic information would ameliorate this deficiency. Common proteins in C.c.p. may have changed enough to preclude homology searches from matching above threshold. A cursory glance at Table 1 indicates that less than 10% of the total peptides detected were matched above threshold using a variety of algorithms. The unmatched peptides may include the most interesting and physiologically important proteins concerning C.c.p.’s overwintering ability. Another shortcoming to our approach is GO’s shortcoming, bias toward well studied proteins. GO is a compendium of well studied proteins deposited by research scientists and curated by GO personnel. Generally, well studied proteins constitute the GO database, with model proteins possessing numerous classifications across the three broad categories. Less studied proteins may only have one classification while other proteins will not even appear. This may lead to erroneous conclusions.

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For example, our GO analysis indicated actin 2 was found only in Winter NDSC larvae (Table 3). However, a manual inspection of our data shows that actin 2 is also found in the Summer larvae (Table 5). Though GO has shortcomings, we believe it is a useful tool in proteomics. MS/MS proteomics allows for the unbiased characterization of the entire protein set in an organism at a particular point in time. Though we did not match the majority of detected peptides, we were able to positively identify over 100 proteins in each sample. To our knowledge, this represents the largest number of proteins identified in a high throughput proteomics experiment on a nonmodel, cold tolerant organism. Significant findings in differential expression between samples include an up-regulation in Winter NDSC of the following GO categories; myosin complex, structural constituent of cytoskeleton, amine metabolic process, gene silencing, and immune system process. Up-regulation of these categories indicates that Winter NDSC rearrange their cytoskeleton relative to Summer larvae, increase amine metabolism, silence gene expression, and increase immune-related proteins. Concerning up-regulated GO categories in Summer, these were numerous including developmental process, general metabolism, transporter activity, and glycoprotein metabolism. Not surprisingly, such categories indicate the Summer larvae are active, foraging, developing, and feeding relative to the senescent Winter NDSC larvae. Especially interesting proteins up-regulated in Winter NDSC larvae include unique cuticle proteins (larval/pupal rigid cuticle protein 66 precursor and larval cuticle protein A2B), and lysozyme C precursor (immune related). Additionally, numerous isoforms of actin and myosin were unique to Winter NDSC while α actinin, tubulin, and tropomyosin were all up-regulated in the Summer larvae, further indicating a cytoskeletal rearrangement.

’ ASSOCIATED CONTENT

bS

Supporting Information Supplemental tables and figures. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Galvin Life Sciences, Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556 mcarrasc@ nd.edu Phone 574-631-9499 Fax 574-631-7413.

’ ACKNOWLEDGMENT We thank Kent Walters for assistance with collecting insects and John Tan, PhD at the Genomics Core Facility at Notre Dame for his help processing and analyzing data. We acknowledge Eksigent Technologies for use of the nanoLC-2D system. This work was supported by NSF grants IOS-0618342 and IOS-1025929. ’ REFERENCES (1) Bennett, V. A.; Sformo, T.; Walters, K.; Toien, O.; Jeannet, K.; Hochstrasser, R.; Pan, Q.; Serriani, A. S.; Barnes, B. M.; Duman, J. G. Comparative overwintering physiology of Alaska and Indiana populations of the beetle Cucujus clavipes (Fabricius): roles of antifreeze proteins, polyols, dehydration and diapause. J. Exp. Biol. 2005, 208, 4467–77. 4645


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