28
Aug

 

 

 

 

 

 

Project No. PC16-980.001

Snapshot Proteomics applied towards pharmacodynamic biomarker discovery:

A pilot study inex-vivo-treated human sera

 

 

Final Report Submitted to Drs. XXXXXXXXXX and XXXXXXXX

11 January 2016

 

 

 

 

AVMBioMed, LLC

33W.KingSt.#181 Malvern, PA19355

Contacts:

 

ChristianLoch, M.P.H, Ph.D.

loch@avmbiomed.com

Phone:(610)846.286.2466×707

Fax: (610) 846.286.2466

Director of Research & Development

 

MauraBuckley

buckley@avmbiomed.com

Phone: (610)846.286.2466×700

Fax: (610) 846.286.2466

BusinessManager

 

This report has been abbreviated and names obscured to protect the scientific integrity and property (i.e.information) of the client. Copyright © 2017 AVMBioMed, LLC

1. Summary

A pilot study was conducted to evaluate the suitability of Snapshot Proteomics towards pharmacodynamic (PD) biomarker discovery for an investigational drug, henceforth known as XXXXX- Compound of Interest or BCOI. All information regarding this compound was and remains blinded to AVMBioMed, except that it inhibits an enzyme in the prostaglandin synthesis pathway and was administered to human sera ex-vivo at 0.082mg/ml. Scientists at XXXXX were responsible for design and implementation of all protocols upstream of the array-based assay (i.e. sample selection, collection, processing, and storage). Dr. Christian Loch, Director of Research and Development, AVMBioMed, was responsible for designing, conducting, and analyzing the assay of enzymatic snapshots.
Results from this study indicate an unqualified success. Optimal assay conditions specific to BCOI were revealed, which will guide future experimental design. Importantly, specific PD biomarkers of BCOI were identified, putative until confirmed in larger validation study: namely, phospho-UCN3, phospho-GAGE1, and ubiquitylated-SEPT6.

 

 

2. Results

The four main treatment groups and the samples evaluated under each were as follows:

  • B1) Serum assayed on arrays in minimal assay buffer with no enzymes or chemical inhibitors added. This condition was used with sample 11 (non-fasting male) and the pool of all 12 samples
  • B2) Serum assayed on arrays in buffer 1 supplemented with cellular energy. This condition was tested with samples 1 and 12 (fasting female, and non-fasting female, respectively)
  • B3) Serum assayed on arrays in buffer 2 supplemented with chemical inhibitors useful to broadly boosting activity of PTM-conjugating enzyme generally. This condition was tested with samples 2 and 3 (fasting female, and fasting male, respectively)
  • B4) Serum assayed on arrays in buffer 3 supplemented with compounds known to enhance specific forms of PTM. This condition was tested with samples 4, 5 and 10 (non-fasting female, non-fasting male, and fasting female, respectively)

As is obvious from the description, these buffers range from most natural (B1) to most manipulated (B4); a more useful classification, however, might sharply delineate [B1 and B2] as distinct from [B3 and B4] based on the use of chemical inhibitors known to influence enzymatic activities, used only in the latter two buffers.
A fifth condition involved utilizing a microarray whose proteins were sourced from a different host system. The advantage mandating it’s testing was larger proteomic coverage; the downside was increased evolutionary distance between the protein production host (not-mammalian) and the serum we tested (human). That array product was found inferior for this particular application, and we will not be discussing data from this condition (used to test samples 6, 7, 8 and 9).

The complete list of proteins whose ubiquitylation or phosphorylation status changed under each of the four conditions can be found in Appendix 1. As shown in Table 1, the number of statistically significant changes (BCOI treated versus control; p<0.05) varied among the conditions tested. However, because the number of samples tested under each condition was not homogeneous, it is important to consider the false discovery rates (FDR) calculated for each list. Based on these values, the number of statistically real changes was determined, and shows that B4 was numerically inferior by wide margin. Interestingly, the second-most artificial buffer examined (B3) resulted in the most changes identified. When the most

“natural” conditions were compared (i.e., B1 and B2), we see almost as many phosphorylation changes as with buffer B3 (n=63 and 60, compared to 70). In the case of ubiquitylation, however, we observe B3- comparable changes only in buffer B2 (n=70 compared with 81).

 

Found FDR Real
B1 PO4 69 8.15% 63
B2 PO4 66 8.52% 60
B3 PO4 76 7.40% 70
B4 PO4 18 0.56% 18
B1 UB 45 12.56% 39
B2 UB 76 7.40% 70
B3 UB 87 6.47% 81
B4 UB 9 1.11% 9

Table 1. Numbers (n) of proteins found to have significantly (p<0.05) changed in phosphorylation (PO4) and ubiquitylation (UB) by treatment (B1-4), alongside the false discovery rate (FDR) associated with each list, and the subsequent number of “real” changes remaining after accounting for that rate.

 

Gene Ontology (GO) clustering can be useful for filtering meaningful changes from false positives and providing insight into the biological response to compound. This process involves taking a specified set of data and asking whether there are any biological processes, molecular functions, or cellular locations over-represented in the data relative to what chance alone would predict. One major caveat, of course, is the incompleteness of the ontology database; information about those three categories is simply not known for all proteins (or poorly annotated even when it is). Nevertheless, it can provide a useful tool to aid in the process of prioritization of hits for follow-up study. It is up to the investigator whether or not to assign higher priority to individuals comprising an ontology based on her own insight and intuition. Ontology analysis was undertaken for changes observed under each condition. Lists were compared against the goa_human database with Benjamini correction for multiple testing (Beissbarth and Speed); results are presented in Appendix 2. Examining these ontologies reveals interesting and intuitive discoveries in all four conditions examined:

  • In the case of B1, interesting ontologies present within the phosphorylation changes included various kinase families (GO:4693 and GO:43405), signal transduction cascades (GO:42770, GO:19933 and GO:7188) and responses to foreign entity (GO:9615). Ubiquitylation ontologies included transport across a membrane (GO:65002), secretion (GO:46903) and receptor signaling (GO:5057).
  • In the case of B2, interesting phosphorylation ontologies included neuron projection (GO:43005), dendrite (GO:30425), axon guidance (GO:7411), transmembrane receptor signaling (GO:7178), and kinase activity (GO:4672). Ubiquitylation ontologies included immune response (GO:6955), hemopoisis (GO:30097), signal transduction (GO:7165), and various serum proteome-specific ontologies (GO:46649, GO:45321, GO:30217).
  • In the case of B3, there were many, often overlapping ontologies revealed for phosphorylation. Interesting ontologies included post-translational modification (GO:43687), NfkB cascade (GO:7249), and several ontologies specific to the serum proteome (GO:50870, GO:46649, GO:46651, GO:45321). Interesting ubiquitylation ontologies were IL6-biosynthesis, and post- translational modification (GO:42226 and GO:43687, respectively). N.b., the list of 12 proteins comprising the PTM ontology for phosphorylation was completely distinct from the 13 comprising the same ontology from the ubiquitylation data.
  • Even in the case of condition B4 which was previously shown numerically inferior, “sensory perception” (GO:7600) was an ontology revealed within both the phosphorylation and ubiquitylation changes. As observed previously, members were distinct (rtp4, chml, gjb3 for phosphorylation; gnat2 and ercc3 for ubiquitylation).

The lists of statistically significant changes to phosphorylation and ubiquitylation observed for treatment groups B1-B4 is included as attachments to this report, as well as the complete datasets from which the significant changes were culled (https://www.dropbox.com/XXXXXXXXXXXXLINKNOTACTIVE). Results by individual serum sample can also be found at this link.

3. Discussion

AVMBioMed has previously utilized Snapshot Proteomics to study the effects of investigational compounds; we have already validated the assay for use in human serum; and we have shown the assay useful towards discovery of PD biomarkers. Here we demonstrate utility of the assay in human sera treated ex-vivo with compound. This novelty and our lack of information regarding BCOI prior to performing the assays dictated the variety of conditions tested. Based on the distribution of data that showed significantly more changes than predicted by chance alone (see FDR in Table 1), we conclude that the assay was successful under all conditions tested. Success was also determined by the intuitive nature of the ontologies uncovered for an analgesic inhibitor of prostaglandin synthesis (e.g., proteins located in serum and cell membranes, involved in signaling, neurological functioning, etc.).

In terms of evaluating the relative superiority of one or more conditions, the experts on this compound (i.e. XXXXX scientists) should carefully evaluate the changes to phosphorylation and ubiquitylation elicited and determine if any set(s) stand out. Absent additional information, it is the recommendation of AVMBioMed to always utilize the assay in as natural a state as possible. Gaining physiologically relevant insight into a compound (including to find PD biomarkers) demands that 1) the assay function, and 2) the sample has been manipulated as little as possible. Further manipulation doesn’t necessarily ruin the assay, as B4 also appears to have been successful statistically speaking. But at the least, experimental manipulation adds variability to the system; and complex human proteomes are variable enough on their own. Our recommendation, then, would be to utilize B2 for all future array-based experiments involving BCOI. We favor B2 to B1 given the positive influence to ubiquitylation elicited by the addition of energy (ATP) regeneration machinery (see Table 1).

Because of the similarity and relative success of the assay in the more natural buffers (B1 and B2), we asked if any PTM changes observed were common to both sets of conditions. Indeed, we identified phosphorylation of UCN3 (increased after BCOI treatment), phosphorylation of GAGE1 (increased in response to BCOI), and ubiquitylation of SEPT6 (decreased in response to BCOI). Importantly, these changes were observed in the pool of all twelve samples, and all three of the individual samples tested under similar conditions (i.e., lacking additional chemical inhibitors). GAGE1 is a serum antigen involved in cellular defense response (Van den Eynde et al.); UCN3 binds G-coupled receptors in response to stress and is linked to pain via the corticotropin-releasing factor (CRF) system (Hu et al.); SEPT6 is found in synaptic vesicles and at the axon terminus (Smith et al.; Moon et al.).

None of the proteins identified under conditions B1 or B2 changed both phosphorylation and their ubiquitylation; rather, each list was completely distinct. In fact, of the 229 total phosphorylation and 217 total ubiquitylation changes identified cumulatively in all four buffers, only two proteins changed both ubiquitylation and phosphorylation in response to drug. (The proteins were MBNL1 and SDS, and both events occurred in B3.) The fact that there is so little overlap in the identity of proteins that changed in phosphorylation and ubiquitylation demonstrates the fact that these events were real, and not simply the product of “noisy” proteins. By extension, this provides assurance that the overlap seen between individuals within a treatment group was real (that is, additional to FDR which statistically quantifies that intuition).

Application of BCOI resulted in decreased phosphorylation/ubiquitylation at some proteins and increased phosphorylation/ubiquitylation at others. This is consistent with expectations and previous results (AVMBioMed), and reflects the complexity of response to any intervention including even, for example, kinase inhibitors. For purposes of our analysis, we have limited our lists and discussion to directionally common changes (i.e., all samples showed increased phosphorylation, or all samples showed decreased phosphorylation). There were 42 total proteins whose phosphorylation changed statistically (p<0.05) in both samples assayed in buffer B4, but only eighteen were directionally common, for example (Table 1). It is not known, nor is it obvious, that expecting PTM changes to occur in similar direction improves the biological validity of resulting data. That is because protein modification exists as a spectrum, never an “all” or “nothing”; and the diversity of human proteomes (reflecting the diversity of genomics, behavior, diet, activity, disease presence, sample collection protocols, etc.) guarantees that two individuals will have different baseline levels of kinases and E3 ligases, as any other protein. If, for example, the administration of drug demands a cellular response mediated by a 40% level of phosphorylation at Protein-X, and baseline phosphorylation levels of Protein-X in two individuals were 20% and 60% respectively, then the differing direction of phosphorylation change observed as both individuals re-set to 40% would indeed have biological meaning. The destination would be more important than the trip, metaphorically speaking. Nevertheless, for purposes of this proof-of-concept pilot study we limited our statistics, ontology analysis and discussion to the more intuitive scenario in which modification changes occurred in common direction among all samples comprising a treatment group.

Not all of the biomarkers suggested must be present in serum to be useful, although all may be. GAGE1, for example, is thought to be silent in adult tissue except testis. If true, that renders its increased phosphorylation unlikely to be a useful biomarker for females as measured in their sera. However, this assay is based on the fact that a particular kinase was present in sera that acted upon recombinant GAGE1 present on the array. The identity of the kinase would be scientifically interesting to know, but it is the activity signature of that kinase that is most relevant. By way of example, consider the utility of
CA-125 as a biomarker, and the lack of basic science knowledge surrounding it.

Sample assignment to each condition (B1-B4) was random, as all information about the compound and the samples was blinded to AVMBioMed until after experiments were conducted. While the assay appears to have been robust across fast/ non-fast conditions, there is no reason to introduce unnecessary experimental variability to an assay that is both highly sensitive and so information rich. Based on the success of this pilot study, it is our recommendation to establish homogenous collection protocols and utilize common assay conditions to validate and identify markers of BCOI efficacy. Those markers suggested by this preliminary study would be evaluated on larger scale, but our assay would remain open to novel discovery across thousands of proteins in the event that biological administration,

In summary, this pilot study was an unqualified success. Optimal assay conditions specific to BCOI were revealed, and specific PD biomarkers were identified- phospho-UCN3, phospho-GAGE1, and ubiquitylated-SEPT6.

 

4. Methods

Human sera was collected from 12 individuals under various protocols (e.g., fasting and non-fasting conditions), and each split into two aliquots. One aliquot was treated with BCOI (18.25mcg/ml), the other mock treated. A pool of all twelve samples was made and handled identically, for a total of thirteen serum samples analyzed for activity Snapshots across twenty-six protein microarrays (13 samples * two treatment arms). Activity snapshots were captured for ubiquitylation and phosphorylation changes in four different conditions 1) serum assay buffer with no enzymes or chemical inhibitors added (proprietary formulation), 2) the same buffer 1 but supplemented with cellular energy machinery 3) buffer 2 supplemented with chemical inhibitors useful to broadly boosting activity of PTM enzymes generally, and 4) buffer 3 supplemented with compounds known to enhance specific forms of PTM. A fifth condition tested involved using a different form of protein microarray that differed most importantly in the host utilized for production of the arrayed proteins.

Protein microarrays were removed from -20oC storage and placed at room temperature (RT) for 15
minutes before opening, to avoid formation of condensation. Arrays were then blocked for 1 hour at RT in PBS containing 0.05% Tween-20, 20mM reduced glutathione, 1mM DTT, 3% BSA, and 25% glycerol. Three PBS washes preceded 90 minutes (RT) incubation with serum supplemented with assay buffer as described. Arrays were washed in PBST (0.05% Tween 20) and two changes of PBS before incubating for 60 minutes with detection reagents specific to ubiquitylation and phosphorylation. Arrays were then washed as before prior to incubation with Cy-conjugated secondary detection reagents. A final wash of 2 changes PBST, 2 x PBS, then two changes water preceded centrifugal drying (1000 RPM for 5 minutes at RT) and scanning (GenePix 4100A by Molecular Devices) of the arrays.

Microarray images were gridded and quantitated using GenePix Pro (v7) software. Median intensities (features and local backgrounds) were utilized, and signal to noise ratio (SNR) calculated. Values were then normalized to biological controls within each array. Duplicate features (representing identical protein) were summarized by average and standard deviation. These values were compared between arrays (compound treated versus mock treated) then Loess transformed by print tip and location to remove technical sources of error (Smyth and Speed 2003), resulting in the final estimate of magnitude change (M-value). T-test (paired, 2 tailed) was used to assess the statistical significance (p-value) of each estimate (under the null hypothesis that M = 0). A threshold of 95% confidence (p < 0.05) was employed to filter data. Gene Ontology (GO) clustering was performed (Beissbarth and Speed) to identify categories (biological processes, cellular components, or molecular functions) over-represented within this data set relative to what chance-alone would predict. N.b., M-value is a twice normalized (biologically and for technical sources of error) difference between mean signal-to-noise ratios generated from relative fluorescence units (RFU); as such it has no units, and can be simply reported or graphed as “M-value”. For simplicity, “RFU” could also be used.

 

7. References

  1. Beissbarth, T. and T.P. Speed (2004). “GOstat: Find statistically overrepresented Gene Ontologies within a group of genes.” Bioinformatics 20(9):1464-1465.genes coding for an antigen recognized by autologous cytolytic T lymphocytes on a human melanoma. J Exp Med. 182(3):689-98.
  2. Van den Eynde B, Peeters O, De Backer O, Gaugler B, Lucas S, Boon T (1995) A genes coding for an antigen recognized by autologous cytolytic T lymphocytes on a human melanoma. J Exp Med. 182(3):689-98.
  3. Hu J, Bai X, Bowen JR, Dolat L, Korobova F, Yu W, Baas PW, Svitkina T, Gallo G, Spiliotis ET. (2012) Septin-driven coordination of actin and microtubule remodeling regulates the collateral branching of axons. Curr Biol 22(12):1109-15.
  4. Smith ML, Li J, Ryabinin AE (2015). Increased alcohol consumption in urocortin 3 knockout mice is unaffected by chronic inflammatory pain. Alcohol Alcohol 50(2):132-9.
  5. Moon IS, Lee H, Walikonis RS (2013). Septin 6 localizes to microtubules in neuronal dendrites. Cytotechnology 65(2):179-86.
  6. Smyth, G. K. and T. Speed (2003). “Normalization of cDNA microarray data.” Methods 31(4): 265-273.

 

8. Explanation of column headers:

Block, row, column = the physical location of the protein listed; block, column, and row of the microarray (there are 48 blocks left to right top to bottom, each with 32 columns and 31 rows).

Name = Protein name
ID = accession number
Mvalue = duplicate-summarized, loess normalized, signal to noise ratio from the protein, values obtained from the experimental array minus those obtained from the negative control array.
Stdev = standard deviation of the difference above (standard deviation of two numbers subtracted is the square root of the sum of squares of the standard deviations associated with each of the two numbers) Note that although calculation of standard deviation technically requires three values, Excel will finesse a value from just two and report it, which is what we are using here (since each protein was only printed in duplicate for spatial limitations)
Pval = p value from 2-tailed, paired T-test for significance of change (with cutoff set at p<0.05).

 

9. Files attached (bold words present in corresponding file names):

  1. All data obtained for ubiquitylation and phosphorylation from each of the serum samples successfully tested.
  2. Statistically significant changes in these samples only.
  3. Results by treatment groups (B1-B4), statistically significant (p<0.05) and directionally common changes to phosphorylation and ubiquitylation.

Appendix 1. Significant (p<0.05) phosphorylation and ubiquitylation changes observed, by buffer treatment

 

Table of Contents

Buffer 1, Phosphorylation 8
Buffer 1, Ubiquitylation 10
Buffer 2, Phosphorylation 11
Buffer 2, Ubiquitylation 13
Buffer 3, Phosphorylation 14
Buffer 3, Ubiquitylation 16
Buffer 4, Phosphorylation 18
Buffer 4, Ubiquitylation 19
Appendix 2 19
  • B1) minimal assay buffer with no enzymes or chemical inhibitors; sample11 (non-fasting male)and the pool of all 12 samples

B1 Phosphorylation

gene Description Sample11 Pool
BRSK1 BR5serine/threonine5kinase51 2.5252378 1.07268063
KCTD18 BTB/POZ5domainFcontaining5protein5KCTD18 2.47098543 0.76017967
N6AMT1 NF65adenineFspecific5DNA5methyltransferase515(putative)5 0.83076552 0.08667986
GFCSF Recombinant5human5GFCSF 0.4837218 0.31926088
TRIB2 tribbles5homolog525(Drosophila)5(TRIB2) 0.44529386 0.22429373
RIBC1 RIB43A5domain5with5coiledFcoils515(RIBC1) 0.40511095 0.12688007
IFNA17 interferon,5alpha5175(IFNA17) 0.40422745 0.19954891
NDUFB11 Neuronal5protein517.35(P17.3),5mRNA 0.35327507 0.0796287
IGHG1 cDNA5clone5MGC:887965IMAGE:6295732,5complete5cds 0.34978893 0.17904898
MPP5 MAGUK5p555subfamily5member55 0.33444484 0.20405331
SYDE1 Rho5GTPaseFactivating5protein5SYDE1 0.32048121 0.05501032
GAGE1 G5antigen515(GAGE1) 0.24001809 0.01311792
PLAT plasminogen5activator,5tissue5(PLAT),5transcript5variant51 0.23997224 0.03147251
CCDC103 Similar5to5RIKEN54933439F115(LOC388389),5mRNA 0.2388714 0.35097007
IMPA1 inositol(myo)F1(or54)Fmonophosphatase515(IMPA1) 0.22099098 0.3037511
ZNF364 zinc5finger5protein53645(ZNF364) 0.20465994 0.28476579
DKFZp761B1 hypothetical5protein5DKFZp761B1075(DKFZp761B107) 0.19251614 0.20815916
GCGR glucagon5receptor5(GCGR) 0.16568106 0.07611381
RPS14 ribosomal.protein.S14.(RPS14),.transcript.variant.3 0.16247812 0.18361065
NKRF NFCkappaB.repressing.factor.(NKRF) 0.15428245 0.05944205
WDR55 WD.repeatCcontaining.protein.55 0.14291953 1.03763531
CDC42EP1 Cdc42.effector.protein.1 0.1302437 0.28725919
C9orf142 chromosome.9.open.reading.frame.142.(C9orf142) 0.12618833 0.3372613
ARFGAP1 ADPCribosylation.factor.GTPase.activating.protein.1.(ARF 0.11983765 0.0918689
TWF2 twinfilin,.actinCbinding.protein,.homolog.2.(Drosophila).( 0.10164471 0.18743406
STAT6 Signal.transducer.and.activator.of.transcription.6 0.09457038 0.11295579
MYL6 myosin,.light.chain.6,.alkali,.smooth.muscle.and.nonCmus 0.09419673 0.11010609
UCN3 Urocortin.3.(stresscopin),.mRNA.(cDNA.clone.MGC:9697 0.09077559 0.15105278
GSPT2 G1.to.S.phase.transition.2.(GSPT2) 0.08362082 0.04988089
VPS37B Hypothetical.protein.FLJ12750.(FLJ12750),.mRNA 0.0639662 0.17983239
LYRM1 LYR.motif.containing.1.(LYRM1) 0.05748776 0.00812979
ING3 inhibitor.of.growth.family,.member.3.(ING3),.transcript.v 0.03117647 0.03396803
CNIH4 cornichon.homolog.4.(Drosophila).(CNIH4) 0.02376809 0.03209839
GAD2 Glutamate.decarboxylase.2 0.00998203 0.0676597
SYP Synaptophysin 0.00968044 0.13678582
FOXP4 forkhead.box.P4.(FOXP4) 0.00439687 0.0216983
DKFZP686A0 LIM.and.calponin.homology.domains.1.(LIMCH1) -0.0022396 -0.0550287
DNAJC12 DnaJ.(Hsp40).homolog,.subfamily.C,.member.12.(DNAJC1 -0.0095859 -0.0640333
C4orf6 chromosome.4.open.reading.frame.6.(C4orf6) -0.0375526 -0.0798194
CCNK,.CDK9 cyclin.K.and.cyclinCdependent.kinase.9.(CDC2Crelated.kin -0.0418058 -0.114048
FLJ42258 FLJ42258.protein.(FLJ42258),.mRNA -0.0436501 -0.1045903
KIAA1600 KIAA1600.(KIAA1600) -0.0451701 -0.0879504
UBXD1 UBX.domain.containing.1.(UBXD1) -0.0488869 -0.4067307
PRPF4 PRP4.preCmRNA.processing.factor.4.homolog.(yeast).(PR -0.0489739 -0.0369877
MED23 Mediator.of.RNA.polymerase.II.transcription.subunit.23 -0.050372 -0.1181783
BTN2A2 butyrophilin,.subfamily.2,.member.A2.(BTN2A2),.transcri -0.0568499 -0.0119045
PLLP plasma.membrane.proteolipid.(plasmolipin).(PLLP) -0.0807333 -0.2422278
CDK6 cyclinCdependent.kinase.6.(CDK6) -0.0904396 -0.095687
WDR1 WD.repeat.domain.1.(WDR1),.transcript.variant.2 -0.095223 -0.102483
MAX MYC.associated.factor.X.(MAX) -0.0972838 -0.0287625
IRF5 interferon.regulatory.factor.5.(IRF5),.transcript.variant.2 -0.0995103 -0.0925117
HSPBAP1 HSPB.(heat.shock.27kDa).associated.protein.1,.mRNA.(cD -0.1011557 -0.0372524
RTN3 reticulon.3.(RTN3),.transcript.variant.1 -0.1156826 -0.1974368
PELI2 pellino.homolog.2.(Drosophila).(PELI2) -0.1195299 -0.2453538
KCNIP4 Kv.channel.interacting.protein.4.(KCNIP4),.transcript.vari -0.1282647 -0.0106148
ABL1 vCabl.Abelson.murine.leukemia.viral.oncogene.homolog.1 -0.1434518 -0.1076897
YY1 YY1.transcription.factor.(YY1) -0.149524 -0.2235959
S100A11 S100.calcium.binding.protein.A11.(S100A11) -0.1712055 -0.2355226
NME6 nonCmetastatic.cells.6,.protein.expressed.in.(nucleosideC -0.1830734 -0.0671742
OXA1L Mitochondrial1inner1membrane1protein1OXA1L -0.1881228 -0.032767
LAS1L LAS16like1(S.1cerevisiae)1(LAS1L) -0.1889549 -0.036333
ETFB electron6transfer6flavoprotein,1beta1polypeptide1(ETFB),1t -0.2657279 -0.5012449
ABL1 v6abl1Abelson1murine1leukemia1viral1oncogene1homolog11 -0.3105311 -0.0053502
CRYBB3 Beta6crystallin1B3 -0.3563187 -0.4861601
DTD1 D6tyrosyl6tRNA(Tyr)1deacylase11 -0.3654193 -0.2913633
PPP2R5E protein1phosphatase12,1regulatory1subunit1B’,1epsilon1isof -0.4772038 -0.0778424
DOCK8 Dedicator1of1cytokinesis1protein18 -1.0814901 -0.3606386
IL5RA Interleukin151receptor,1alpha1(IL5RA),1transcript1variant13, -1.1083297 -0.228027
DUSP16 dual1specificity1phosphatase1161(DUSP16) -1.2150991 -0.2127852

 

B1 Ubiquitylation

gene Description Sample11 Pool
NUP50 nucleoporin750kDa7(NUP50),7transcript7variant72 1.10730082 0.87191575
FMNL2 ForminFlike7protein72 0.71278376 0.49046679
LOC284422 PREDICTED:7Homo7sapiens7similar7to7HSPC3237(LOC2844 0.58826053 0.92953853
SPANXB1 SPANX7family,7member7B17(SPANXB1) 0.46587964 0.26441293
C1orf142 chromosome717open7reading7frame71427(C1orf142) 0.44807042 0.37902747
MB myoglobin7(MB),7transcript7variant71 0.4278984 0.08373015
FHL3 four7and7a7half7LIM7domains737(FHL3) 0.40645294 0.3954461
TGDS TDPFglucose74,6Fdehydratase7(TGDS) 0.35873294 0.20316336
RBPMS RNA7binding7protein7with7multiple7splicing7(RBPMS) 0.31202756 0.05493186
C5orf3 chromosome757open7reading7frame737(C5orf3) 0.28471298 0.36764529
TPM4 tropomyosin747(TPM4) 0.27960759 0.14273801
HSD17B8 hydroxysteroid7(17Fbeta)7dehydrogenase787(HSD17B8) 0.1733529 0.09775779
CDC14B CDC147cell7division7cycle7147homolog7B7(S.7cerevisiae)7(CD 0.15835491 0.57990424
C4orf35 chromosome747open7reading7frame7357(C4orf35) 0.15518681 0.27116361
ACSL4 acylFCoA7synthetase7longFchain7family7member747(ACSL4) 0.14791328 0.12036645
FLJ22028 pyridine7nucleotideFdisulphide7oxidoreductase7domain717 0.12495741 0.19262617
KIAA1147 KIAA11477(KIAA1147) 0.11869554 1.25809787
SYCN PREDICTED:7Homo7sapiens7syncollin7(SYCN) 0.11790377 0.00764191
PIM1 pimF17oncogene7(PIM1) 0.11457539 0.5278955
ARNT aryl7hydrocarbon7receptor7nuclear7translocator7(ARNT) 0.11396009 0.01848019
MTMR9 myotubularin7related7protein797(MTMR9) 0.11010678 0.23988931
ADM adrenomedullin7(ADM) 0.10965952 0.47479744
CCDC110 KMFHNF17protein,7mRNA7(cDNA7clone7MGC:433957IMAGE 0.10031091 0.02451026
CKLF ChemokineFlike7factor7(CKLF),7transcript7variant73,7mRNA 0.02934911 0.46789623
ATP5C1 ATP7synthase,7H+7transporting,7mitochondrial7F17comple 0.01774612 0.21959777
MGC87631 similar7to7hypothetical7protein7FLJ364927(MGC87631) 0.00476233 0.43671087
TBC1D21 TBC17domain7family,7member7217(TBC1D21) 0.00037997 0.2118895
C1orf119 chromosome717open7reading7frame71197(C1orf119) -0.0171386 -0.0798658
PPIA peptidylprolyl7isomerase7A7(cyclophilin7A)7(PPIA),7transcri -0.0275712 -0.0940791
6-Sep septin767(SEPT6),7transcript7variant7V -0.0306431 -0.2406168
MAP3K14 mitogen/activated4protein4kinase4kinase4kinase414 -0.0557374 -0.2962225
OXSR1 Serine/threonine/protein4kinase4OSR1 -0.101398 -0.1514088
GALNT13 Polypeptide4N/acetylgalactosaminyltransferase413 -0.1110697 -0.7129596
LOC387816 Coiled/coil4domain/containing4protein415 -0.1171767 -0.2134505
TFPT TCF34(E2A)4fusion4partner4(in4childhood4Leukemia)4(TFPT) -0.1511065 -0.1848702
DDAH2 dimethylarginine4dimethylaminohydrolase424(DDAH2) -0.1813864 -0.1406148
KLK11 Kallikrein4114(KLK11),4transcript4variant42,4mRNA -0.1821799 -0.2601541
LINC00544 Novel4protein4(Similar4to4bA90M5.1)44[Source:UniProtKB/ -0.2036441 -0.1645776
GM/CSF Granulocyte/macrophage4colony/stimulating4factor -0.2294615 -0.3331045
SENP2 SUMO1/sentrin/SMT34specific4peptidase424(SENP2) -0.2630397 -0.9244414
GAS2 growth4arrest/specific424(GAS2),4transcript4variant42 -0.5179415 -0.5222052
TIGD1 Tigger4transposable4element/derived4protein41 -0.6136727 -0.378767
PGDS prostaglandin4D24synthase,4hematopoietic4(PGDS) -1.3426611 -1.2209529
CUTC cutC4copper4transporter4homolog4(E.4coli)4(CUTC) -2.0924328 -0.5254921
C2orf53 Uncharacterized4protein4C2orf53 -4.043584 -2.1130905

 

  • B2) buffer 1 supplemented with cellular energy; samples 1 and 12 (fasting female, and non- fasting female, respectively)

B2 Phosphorylation

gene Description Sample1 Sample12
PDE7B phosphodiesterase87B8(PDE7B) 1.47591656 0.61404972
KIAA1191 UPF04988protein8KIAA1191 1.25406482 3.93099099
ASAP2 Development8and8differentiationJenhancing8factor82 0.71633714 0.39353343
FLJ22222 hypothetical8protein8FLJ222228(FLJ22222) 0.61657797 0.34970762
UNG uracilJDNA8glycosylase8(UNG),8transcript8variant82 0.58078894 0.54646028
PBK PDZ8binding8kinase8(PBK) 0.52354867 0.59996656
TIA1 TIA18cytotoxic8granuleJassociated8RNA8binding8protein8(T 0.47226068 0.50369698
DPYSL5 DihydropyrimidinaseJrelated8protein85 0.37825026 0.26966837
SNRPB small8nuclear8ribonucleoprotein8polypeptides8B8and8B18(S 0.37427141 0.79824407
FHIT fragile8histidine8triad8gene8(FHIT) 0.34787301 0.22726735
NUBPL nucleotide8binding8proteinJlike8(NUBPL) 0.3175635 0.0909467
GAGE1 G8antigen818(GAGE1) 0.31741115 0.56248947
PSMB3 proteasome8(prosome,8macropain)8subunit,8beta8type,838 0.30481603 0.37915297
UCN3 UrocortinJ3 0.27194719 0.27868671
SMURF1 SMAD8specific8E38ubiquitin8protein8ligase818(SMURF1),8tra 0.15662201 0.56645083
MELK a8serine/threonione8kinase 0.15204682 0.10168284
BTN1A1 Butyrophilin8subfamily818member8A1 0.14848509 0.93232896
CDCA7L Cell8division8cycleJassociated87Jlike8protein 0.11781441 0.07583372
ZNF287 Zinc8finger8protein82878(ZNF287),8mRNA 0.10603307 0.07696778
ANK1 ankyrin+1,+erythrocytic+(ANK1) 0.10246998 0.05573248
DYNC2LI1 dynein,+cytoplasmic+2,+light+intermediate+chain+1,+mRNA+ 0.0961586 0.03487968
CDK5R1 cyclinNdependent+kinase+5,+regulatory+subunit+1+(p35)+(CD 0.07390018 0.26631549
ZNF239 zinc+finger+protein+239+(ZNF239) 0.06702 0.18077746
WDR92 Hypothetical+protein+BC014022,+mRNA+(cDNA+clone+MGC 0.06497388 0.0978798
SH3GL2 SH3Ndomain+GRB2Nlike+2+(SH3GL2) 0.06127151 0.02156319
SESN3 sestrin+3+(SESN3) 0.06041059 0.16487963
CD63 CD63+/+TspanN30+/+TetraspaninN30+Protein+(His+Tag) 0.05563792 0.0242336
YPEL2 Protein+yippeeNlike+2 0.05018718 0.16006376
DLEU1 LeukemiaNassociated+protein+1 0.0462298 0.16646569
MAPK10 MitogenNactivated+protein+kinase+10 0.04156465 0.10400938
KLHDC3 Kelch+domain+containing+3+(KLHDC3),+mRNA 0.03543177 0.23768293
ATF1 activating+transcription+factor+1+(ATF1) 0.03065252 0.14515811
VEGF VEGF+Recombinant+Human+Protein 0.03010375 0.07402113
C1orf109 chromosome+1+open+reading+frame+109+(C1orf109) 0.02488857 0.03039597
PF4V1 platelet+factor+4+variant+1+(PF4V1) 0.01769364 2.23253573
SYT1 synaptotagmin+I+(SYT1) -0.0051039 -0.0856927
LOC284297 Scavenger+receptor+cysteineNrich+domainNcontaining+prot -0.0090858 -0.2615679
SUB1 SUB1+homolog+(S.+cerevisiae)+(SUB1) -0.0115049 -0.0002441
RAB13 RAB13,+member+RAS+oncogene+family+(RAB13) -0.0220872 -0.0552125
SLAMF7 SLAMF7+/+CRACC+/+CD319+Protein+(His+Tag) -0.0303245 -0.0867955
LRRTM4 leucine+rich+repeat+transmembrane+neuronal+4+(LRRTM4) -0.0331277 -0.5698375
COX5B cytochrome+c+oxidase+subunit+Vb+(COX5B) -0.0337032 -0.1018428
RBP5 retinol+binding+protein+5,+cellular+(RBP5) -0.0349877 -0.240446
MYLK Myosin+light+chain+kinase,+smooth+muscle -0.0365316 -0.0509992
MRPL42 mitochondrial+ribosomal+protein+L42+(MRPL42),+nuclear+g -0.0494814 -0.3253569
MAPK10 mitogenNactivated+protein+kinase+10+(MAPK10),+transcrip -0.0559268 -0.2246758
EPS8L2 EPS8Nlike+2+(EPS8L2) -0.0568435 -0.0382335
VPS29 vacuolar+protein+sorting+29+homolog+(S.+cerevisiae)+(VPS2 -0.0627511 -0.0768047
GTPBP6 Putative+GTPNbinding+protein+6 -0.0672492 -0.1305411
BRUNOL4 brunoNlike+4,+RNA+binding+protein+(Drosophila)+(BRUNOL -0.0681506 -0.5237166
PARVA parvin,+alpha+(PARVA) -0.0763217 -0.1079367
AAAS achalasia,+adrenocortical+insufficiency,+alacrimia+(Allgrov -0.0770454 -0.019587
IGHG1 Immunoglobulin+heavy+constant+gamma+1+(G1m+marker) -0.0795739 -0.012861
MAP3K7IP1 mitogenNactivated+protein+kinase+kinase+kinase+7+interact -0.0805317 -0.0919557
EDF1 endothelial+differentiationNrelated+factor+1+(EDF1),+transc -0.0831251 -0.1660386
RSAD1 radical+SNadenosyl+methionine+domain+containing+1+(RSA -0.0921498 -1.9826477
GAS7 Growth+arrestNspecific+7+(GAS7),+transcript+variant+c,+mRN -0.0971154 -0.3012503
KIAA0415 KIAA0415+(KIAA0415) -0.1088582 -0.075569
SFRS9 splicing+factor,+arginine/serineNrich+9+(SFRS9) -0.1156299 -0.1510263
DUSP14 dual+specificity+phosphatase+14+(DUSP14) -0.1314244 -0.0795472
MED4 Mediator+of+RNA+polymerase+II+transcription+subunit+4 -0.1423271 -0.1371331
ZBED2 zinc+finger,+BEDNtype+containing+2+(ZBED2) -0.14394 -0.0927599
PLVAP plasmalemma+vesicle+associated+protein+(PLVAP) -0.146415 -0.0631887
TMEM166 transmembrane+protein+166+(TMEM166) -0.1570864 -0.0831918
ZNF765 zinc+finger+protein+765+(ZNF765) -0.1782994 -0.1761764
ULK4 Serine/threonineNprotein+kinase+ULK4 -0.2293348 -0.6057273

B2 Ubiquitylation

gene Description Sample1 Sample12
ZNF207 zinc8finger8protein82078(ZNF207),8transcript8variant82 1.12661744 2.78485369
HMGA1 high8mobility8group8ATNhook818(HMGA1),8transcript8varia 1.08269473 0.59432413
P4HA2 Prolyl84Nhydroxylase8subunit8alphaN2 1.02211678 0.99887656
TAOK3 TAO8kinase838(TAOK3) 0.89165749 0.1007518
MAP4K2 mitogenNactivated8protein8kinase8kinase8kinase8kinase828( 0.82176556 0.11999072
LCK lymphocyteNspecific8protein8tyrosine8kinase8(LCK) 0.77545584 0.21743913
ZBTB8OS zinc8finger8and8BTB8domain8containing888opposite8strand8 0.67489503 0.17619593
IARS isoleucylNtRNA8synthetase8(IARS) 0.55353003 1.3110905
IL9 interleukin898(IL9) 0.52373565 0.23119341
MREG Melanoregulin 0.47306254 0.38178083
ICA1 Islet8cell8autoantigen81,869kDa8(ICA1),8transcript8variant83 0.4724196 0.3359346
FXR8LBD8 Farnesoid8X8Receptor,8Ligand8Binding8Domain8(FXRNLBD) 0.45712747 0.2322639
BID BH38interacting8domain8death8agonist8(BID) 0.39801064 0.19833668
PSME1 proteasome8(prosome,8macropain)8activator8subunit818(P 0.38542572 0.30677703
NIP7 nuclear8import878homolog8(S.8cerevisiae)8(NIP7) 0.37139388 0.11667324
AP3S1 AdaptorNrelated8protein8complex83,8sigma818subunit8(AP3 0.35791372 0.9270309
HBG1 hemoglobin,8gamma8A8(HBG1) 0.32876457 0.6574465
ASB7 ankyrin8repeat8and8SOCS8boxNcontaining878(ASB7),8transc 0.24424052 0.85066141
AICDA activationNinduced8cytidine8deaminase8(AICDA) 0.24172873 0.15478959
MAN2C1 mannosidase,8alpha,8class82C,8member818(MAN2C1) 0.17969969 0.11275217
RNASE8 ribonuclease,8RNase8A8family,888(RNASE8) 0.17296055 0.48459666
C9orf9 chromosome898open8reading8frame898(C9orf9) 0.16191542 1.52787588
FNTB farnesyltransferase,8CAAX8box,8beta8(FNTB) 0.14084212 0.08063765
ASB6 ankyrin8repeat8and8SOCS8boxNcontaining868(ASB6),8transc 0.13035483 0.16065749
SETD4 SET8domain8containing848(SETD4),8transcript8variant82 0.12284886 0.33515161
ZRANB2 zinc8finger,8RANNbinding8domain8containing828(ZRANB2) 0.12123329 0.05856364
FTSJ1 FtsJ8homolog818(E.8coli)8(FTSJ1),8transcript8variant81 0.08941343 0.05247929
RASL12 RASNlike,8family8128(RASL12) 0.06605741 2.94282813
NFYB Nuclear8transcription8factor8Y8subunit8beta 0.06308296 0.18289172
FGFBP1 fibroblast8growth8factor8binding8protein818(FGFBP1) 0.04514645 0.33315173
NPM1 nucleophosmin8(nucleolar8phosphoprotein8B23,8numatri 0.03934155 0.2669435
THEM5 thioesterase8superfamily8member858(THEM5) 0.023532 0.03905861
COL4A3BP collagen,8type8IV,8alpha838(Goodpasture8antigen)8binding8 0.00497489 0.0522496
PDE4D phosphodiesterase84D,8cAMPNspecific8(phosphodiesteras 0.00129058 0.71919067
IL2RB IL2RB8Protein -0.0301162 -0.0549768
RNF113A ring8finger8protein8113A8(RNF113A) -0.0316966 -0.1328562
GALE UDPNgalactoseN4Nepimerase8(GALE),8transcript8variant81 -0.0453967 -1.5197194
NGLY1 NNglycanase818(NGLY1) -0.0726424 -0.064575
DNAJC16 DnaJ8(Hsp40)8homolog,8subfamily8C,8member8168(DNAJC1 -0.1564448 -0.4986265
KLHL17 kelchNlike8178(Drosophila)8(KLHL17) -0.1817347 -0.0298711
CAPS calcyphosine8(CAPS),8transcript8variant81 -0.2070885 -0.0767833
ELA2B elastase82B8(ELA2B) -0.224612 -0.0814033
6-Sep septin868(SEPT6),8transcript8variant8II -0.2310396 -0.1594657
AFG3L1 AFG38ATPase8family8gene83Nlike818(S.8cerevisiae)8(AFG3L1) -0.2733023 -0.1781789
EPM2A epilepsy,8progressive8myoclonus8type82A,8Lafora8disease8 -0.274474 -0.0778108
TMEM176A transmembrane0protein0176A0(TMEM176A) -0.2840694 -0.0550604
GDAP1 ganglioside6induced0differentiation6associated0protein010 -0.2860004 -0.3032232
PCK1 phosphoenolpyruvate0carboxykinase010(soluble)0(PCK1) -0.2997708 -0.0101608
PVRL1 poliovirus0receptor6related010(herpesvirus0entry0mediato -0.3059865 -0.0845475
SRPX sushi6repeat6containing0protein,0X6linked0(SRPX) -0.3184455 -0.3640709
C21orf37 Chromosome0210open0reading0frame0370(C21orf37),0mRN -0.3198364 -0.164539
PLA1A phospholipase0A10member0A0(PLA1A) -0.3220995 -0.0393013
IL2 Interleukin62 -0.3331258 -0.2563096
FGD6 FYVE,0RhoGEF0and0PH0domain0containing060(FGD6) -0.3337524 -0.2477312
C22orf13 chromosome0220open0reading0frame0130(C22orf13) -0.3468164 -0.0289673
CLDN2 claudin020(CLDN2) -0.4308619 -0.570621
MRPL21 39S0ribosomal0protein0L21,0mitochondrial -0.4428415 -0.293955
TCF25 Transcription0factor025 -0.4625157 -0.4721927
RPL37A ribosomal0protein0L37a0(RPL37A) -0.4662074 -0.6309104
KIR2DL3 Killer0cell0immunoglobulin6like0receptor,0two0domains,0lo -0.4931649 -0.015434
XYLB cDNA0clone0IMAGE:6166461,0containing0frame6shift0erro -0.4951175 -0.2540246
LARP6 La0ribonucleoprotein0domain0family,0member060(LARP6),0 -0.4959835 -0.5565428
IFNGR2 interferon0gamma0receptor020(interferon0gamma0transdu -0.5474679 -0.405604
CORO2A Coronin62A -0.5652344 -0.6025785
FBXL2 F6box0and0leucine6rich0repeat0protein020(FBXL2) -0.5662325 -0.017223
FCRLB Fc0receptor0like02,0mRNA0(cDNA0clone0MGC:711410IMAGE -0.5798743 -0.2707744
CX62 Gap0junction0alpha6100protein -0.6381535 -0.0107587
FLJ45256 Putative0uncharacterized0protein0FLJ45256 -0.6583992 -0.1484559
LST1 Leukocyte0specific0transcript010(LST1),0transcript0variant01 -0.6601093 -0.0934195
GTF3C5 general0transcription0factor0IIIC,0polypeptide05,063kDa0(G -0.673246 -0.4313313
MGC24103 hypothetical0protein0MGC241030(MGC24103) -0.6855898 -0.4995922
TMEM177 transmembrane0protein01770(TMEM177),0transcript0varia -0.7343829 -0.2999365
TRPV4 transient0receptor0potential0cation0channel,0subfamily0V,0 -0.8975146 -0.3926146
HMBS hydroxymethylbilane0synthase0(HMBS),0transcript0variant -0.8981822 -0.0622769
MGC21881 Hypothetical0protein0MGC218810(MGC21881),0mRNA -0.9951224 -0.3327195
C10orf65 chromosome0100open0reading0frame0650(C10orf65) -0.4463994 -0.2650138

 

  • B3) buffer 2 supplemented with chemical inhibitors useful to broadly boosting activity of PTM enzymes generally; tested with samples 2 and 3 (fasting female, and fasting male, respectively)

 

B3 Phosphorylation

gene Description Sample2 Sample3
POMZP3 POM1217and7ZP37fusion7protein 0.76387841 0.09123305
MAP3K7 mitogenEactivated7protein7kinase7kinase7kinase777(MAP3K 0.67804321 0.37735443
C19orf33 chromosome7197open7reading7frame7337(C19orf33) 0.26552843 0.25175162
RAPGEF3 Rap7guanine7nucleotide7exchange7factor73 0.26108332 0.1882194
STARD10 StARErelated7lipid7transfer7(START)7domain7containing710 0.22586261 0.3779379
KLHDC4 kelch7domain7containing747(KLHDC4) 0.21857835 0.0265894
DNAI1 Dynein7intermediate7chain71,7axonemal 0.21736495 0.11897209
ZNF434 zinc7finger7protein74347(ZNF434) 0.21292117 0.09224235
SCN1B Sodium+channel+subunit+beta51 0.14372054 0.10965352
SMNDC1 survival+motor+neuron+domain+containing+1+(SMNDC1) 0.13227798 0.12180613
ICK Intestinal+cell+kinase+(ICK),+mRNA 0.1302643 0.18627465
N/A cDNA+clone+MGC:17410+IMAGE:4156035,+complete+cds 0.12787466 0.10190937
NCK1 NCK+adaptor+protein+1+(NCK1) 0.1141159 0.04465961
HM13 histocompatibility+(minor)+13+(HM13),+transcript+variant+ 0.10930069 0.031289
VAX1 Ventral+anterior+homeobox+1+(VAX1),+mRNA 0.10028245 0.00558106
STK24 Serine/threonine5protein+kinase+24 0.09754934 0.13300355
NEDD8 neural+precursor+cell+expressed,+developmentally+down5r 0.09375641 0.05865679
FKBP2 FK506+binding+protein+2,+13kDa+(FKBP2),+transcript+varian 0.09073891 0.19842869
FAM122C family+with+sequence+similarity+122C+(FAM122C) 0.08959353 0.04195562
SFN stratifin+(SFN) 0.08067301 0.25868404
ILVBL ilvB+(bacterial+acetolactate+synthase)5like+(ILVBL),+transcr 0.08055529 0.09583208
LGP2 DEXH+(Asp5Glu5X5His)+box+polypeptide+58+(DHX58) 0.07944192 0.24811878
MGC31957 hypothetical+protein+MGC31957+(MGC31957) 0.07805334 0.18260795
G5CSF Recombinant+human+G5CSF 0.07698472 0.23544618
SDS serine+dehydratase+(SDS) 0.07474421 0.12706235
FAM32A family+with+sequence+similarity+32,+member+A+(FAM32A) 0.06850264 0.17180205
KIAA0947 Uncharacterized+protein+KIAA0947 0.06522747 0.06093329
ZKSCAN3 zinc+finger+with+KRAB+and+SCAN+domains+3+(ZKSCAN3) 0.0625755 0.44292094
MAN2C1 mannosidase,+alpha,+class+2C,+member+1+(MAN2C1) 0.04402015 0.16445028
SUCLG1 Succinyl5CoA+ligase+[GDP5forming]+subunit+alpha,+mitoch 0.03824732 0.09109485
WISP2 WNT1+inducible+signaling+pathway+protein+2,+mRNA+(cDN 0.03398459 0.0538047
OR1A2 Olfactory+receptor,+family+1,+subfamily+A,+member+2,+mR 0.030617 0.08332889
MXI1 MAX+interactor+1+(MXI1),+transcript+variant+1,+mRNA 0.02901942 0.16150207
CLUAP1 clusterin+associated+protein+1+(CLUAP1) 0.02625456 0.02634528
MRPL43 mitochondrial+ribosomal+protein+L43+(MRPL43) 0.02611765 0.10395452
MBNL1 muscleblind5like+(Drosophila)+(MBNL1),+transcript+variant 0.024779 0.11586474
ERVK351 similar+to+envelope+protein+(LOC113386) 0.02405539 0.01716135
DFFA DNA+fragmentation+factor,+45kDa,+alpha+polypeptide+(DF 0.02347879 0.08569834
DTYMK deoxythymidylate+kinase+(thymidylate+kinase)+(DTYMK) 0.02144117 0.03908478
C15orf38 chromosome+15+open+reading+frame+38+(C15orf38) 0.01837835 0.00767966
PUF60 Poly(U)5binding5splicing+factor+PUF60 0.01564448 0.11802603
RPE ribulose555phosphate535epimerase+(RPE) 0.01543654 0.1357843
VPS35 vacuolar+protein+sorting+35+homolog+(S.+cerevisiae)+(VPS3 0.01416142 0.04606923
KIT v5kit+Hardy5Zuckerman+4+feline+sarcoma+viral+oncogene+h 0.01182822 0.19187172
MELK a+serine/threonione+kinase 0.01064383 0.08464762
CD40 CD40+molecule,+TNF+receptor+superfamily+member+5+(CD 0.01034364 0.1017415
ZKSCAN1 zinc+finger+with+KRAB+and+SCAN+domains+1+(ZKSCAN1) 0.00816861 0.30267048
PON3 Serum+paraoxonase/lactonase+3 0.00030347 0.09211751
PCTK3 PCTAIRE+protein+kinase+3+(PCTK3) -183117 -712143
G6PD Glucose565phosphate+15dehydrogenase -246453 -3905097
WBP2 WW+domain+binding+protein+2+(WBP2) -306772 -718759
NAGK N5acetylglucosamine+kinase+(NAGK) -319093 -525258
AFG3L1 AFG3+ATPase+family+gene+35like+1+(S.+cerevisiae)+(AFG3L1) -362978 -158951
PRO1853 Protein+midA+homolog,+mitochondrial -439564 -1015281
CHEK1 CHK1+checkpoint+homolog+(S.+pombe)+(CHEK1) -547247 -3752763
DHRS1 Dehydrogenase/reductase4SDR4family4member41 -0.0572665 -0.1245782
STYX serine/threonine/tyrosine4interacting4protein4(STYX) -0.0590796 -0.007835
LOC388882 hypothetical4LOC3888824(LOC388882) -0.0612278 -0.1730647
LOC286208 PREDICTED:4Homo4sapiens4hypothetical4protein4LOC2862 -0.0813594 -0.0285709
KDELR1 KDEL4(Lys:Asp:Glu:Leu)4endoplasmic4reticulum4protein4re -0.0884883 -0.0293814
AP2M1 adaptor:related4protein4complex42,4mu414subunit4(AP2M -0.0936246 -0.0708953
APEH N:acylaminoacyl:peptide4hydrolase4(APEH) -0.0944639 -0.2480651
GLB1L3 hypothetical4protein4BC0110014(LOC112937) -0.0945873 -0.0049473
SECTM1 secreted4and4transmembrane414(SECTM1) -0.1083732 -0.0974485
SPRYD4 SPRY4domain:containing4protein44 -0.1136889 -0.1878273
C18orf8 chromosome4184open4reading4frame484(C18orf8) -0.1177128 -0.0442938
ALOX12 arachidonate412:lipoxygenase4(ALOX12) -0.1206277 -0.1317215
CNNM1 cyclin4M14(CNNM1) -0.1243219 -0.1732481
SPCS2 signal4peptidase4complex4subunit424homolog4(S.4cerevisia -0.1253471 -0.0668173
TMUB1 transmembrane4and4ubiquitin:like4domain4containing414( -0.1255586 -0.5223248
GTPBP4 GTP4binding4protein444(GTPBP4),4mRNA -0.1259622 -0.0634006
MGAT4A Mannosyl4(alpha:1,3:):glycoprotein4beta:1,4:N:acetylgluc -0.1327114 -0.0730054
MRO Protein4maestro -0.1350217 -0.0687886
ENTPD1 ectonucleoside4triphosphate4diphosphohydrolase414(ENT -0.1701162 -0.3655582
SUV39H2 suppressor4of4variegation43:94homolog424(Drosophila)4(SU -0.1985637 -0.0437229
RPUSD4 RNA4pseudouridylate4synthase4domain4containing444(RPU -0.9166695 -0.025446

 

B3 Ubiquitylation

gene Description Sample2 Sample3
RAD51AP1 RAD517associated7protein717(RAD51AP1) 1.82366503 1.40678907
PSCD2 pleckstrin7homology,7Sec77and7coiledGcoil7domains727(cyt 1.70810345 0.73361822
ITGB3BP Centromere7protein7R 1.54987168 0.03913556
MBNL1 muscleblindGlike7(Drosophila)7(MBNL1) 1.02500612 0.98640233
UTS2 Urotensin727(UTS2),7transcript7variant72,7mRNA 0.96473075 1.97644063
SPON2 spondin72,7extracellular7matrix7protein7(SPON2) 0.95544473 1.44550103
ACYP2 acylphosphatase72,7muscle7type7(ACYP2) 0.94229162 0.55072602
FHL3 four7and7a7half7LIM7domains737(FHL3) 0.78979655 0.40015733
CDC7 Cell7division7cycle77Grelated7protein7kinase 0.6855229 0.82738251
SLC26A1 Sulfate7anion7transporter71 0.64137766 0.50332414
AGR2 anterior7gradient7homolog727(Xenopus7laevis)7(AGR2) 0.54097824 0.59984489
SDHB succinate7dehydrogenase7complex,7subunit7B,7iron7sulfur7 0.50229272 0.56732142
MPP7 membrane7protein,7palmitoylated777(MAGUK7p557subfam 0.48950066 0.11741386
SPRR2G Small7prolineGrich7protein72G 0.48721791 0.56139804
FBXO7 FGbox7protein777(FBXO7),7transcript7variant71 0.46300562 0.53235686
YIPF4 Yip17domain7family,7member747(YIPF4) 0.43744114 0.31993628
BTN2A2 butyrophilin,7subfamily72,7member7A27(BTN2A2),7transcri 0.39044817 0.08114807
AGPAT4 1GacylglycerolG3Gphosphate7OGacyltransferase747(lysophos 0.38483945 0.28328052
SPSB1 splA/ryanodine7receptor7domain7and7SOCS7box7containin 0.36706991 0.89176739
RBM17 Splicing-factor-45 0.35769841 1.54719501
EEF1DP3 Putative-elongation-factor-1BdeltaBlike-protein 0.32419569 0.30167519
C11orf53 chromosome-11-open-reading-frame-53-(C11orf53) 0.29003218 0.02659195
BUB1 BUB1-budding-uninhibited-by-benzimidazoles-1-homolog- 0.28388723 0.45912227
MCPH1 microcephaly,-primary-autosomal-recessive-1-(MCPH1) 0.28184737 0.15412123
TXNL5 thioredoxin-domain-containing-17-(TXNDC17) 0.27249637 0.1320424
SPTB spectrin,-beta,-erythrocytic-(includes-spherocytosis,-clinic 0.26810019 0.13449882
CDRT4 CMT1A-duplicated-region-transcript-4-(CDRT4) 0.25851089 0.7093685
IL1F8 interleukin-1-family,-member-8-(eta)-(IL1F8),-transcript-va 0.25375259 0.57098405
DBC1 deleted-in-bladder-cancer-1-(DBC1) 0.20843512 0.00053455
GNAT2 guanine-nucleotide-binding-protein-(G-protein),-alpha-tra 0.20324961 0.32875347
LOC283034 PREDICTED:-Homo-sapiens-hypothetical-LOC283034-(LOC 0.19013427 0.25467635
GJB3 gap-junction-protein,-beta-3,-31kDa-(GJB3),-transcript-var 0.18430741 0.60656642
GK3P glycerol-kinase-3-pseudogene-(GK3P) 0.18382766 0.11625025
KCNA4 Potassium-voltageBgated-channel,-shakerBrelated-subfam 0.17294492 0.01206345
MPP3 membrane-protein,-palmitoylated-3-(MAGUK-p55-subfam 0.17150725 0.28736467
IL17F Interleukin-17F-(IL17F),-mRNA 0.15913545 0.23852506
IGHG1 Ig-gammaB1-chain-C-region 0.14156123 0.51482414
UROS uroporphyrinogen-III-synthase-(congenital-erythropoietic 0.14057789 0.17829252
UBE2V1 ubiquitinBconjugating-enzyme-E2-variant-1-(UBE2V1) 0.1370734 0.30371692
PKN3 Serine/threonineBprotein-kinase-N3 0.12158388 0.36752514
ZFYVE27 Zinc-finger-FYVE-domainBcontaining-protein-27 0.11994001 0.18777226
UQCR10 UbiquinolBcytochrome-c-reductase-complex-(7.2-kD)-(UCR 0.11806669 0.2335189
C1orf111 chromosome-1-open-reading-frame-111-(C1orf111) 0.10986681 0.19863905
FBXL12 FBbox/LRRBrepeat-protein-12 0.10541342 0.1290207
SESN3 sestrin-3-(SESN3) 0.09523481 0.12171278
SERINC2 serine-incorporator-2-(SERINC2) 0.08757122 0.00456786
IL19 Interleukin-19-(IL19),-transcript-variant-2,-mRNA 0.08481296 0.08342868
KCNS3 potassium-voltageBgated-channel,-delayedBrectifier,-subf 0.07535966 0.14266914
IL2RB IL2RB-Protein 0.06302472 0.08055087
CYB5A cytochrome-b5-type-A-(microsomal)-(CYB5A) 0.05580563 0.03031232
C17orf42 chromosome-17-open-reading-frame-42-(C17orf42) 0.05148554 0.66887743
HSD3B2 hydroxyBdeltaB5Bsteroid-dehydrogenase,-3-betaB-and-ster 0.05048114 0.99387495
TSPAN31 tetraspanin-31-(TSPAN31) 0.04031632 0.72376969
SAT2 spermidine/spermine-N1Bacetyltransferase-2-(SAT2) 0.03111536 0.23767787
CFDP1 craniofacial-development-protein-1-(CFDP1) 0.02824221 0.07762824
SDS serine-dehydratase-(SDS) 0.01182796 0.08897958
CYP4Z1 cytochrome-P450,-family-4,-subfamily-Z,-polypeptide-1-(C 0.00563672 0.16432302
RAP2B RAP2B,-member-of-RAS-oncogene-family-(RAP2B) -0.0113431 -0.1071748
BID BH3-interacting-domain-death-agonist-(BID) -0.0306384 -0.0332323
ROPN1B RopporinB1B -0.0339067 -0.0915477
MB myoglobin-(MB),-transcript-variant-1 -0.0435045 -0.4057524
BID BH3-interacting-domain-death-agonist-(BID) -0.049128 -0.0148487
PRKG1 protein-kinase,-cGMPBdependent,-type-I-(PRKG1) -0.0973695 -0.0950082
TBXAS1 thromboxane-A-synthase-1-(platelet,-cytochrome-P450,-f -0.101709 -0.1142503
GLYATL2 glycineBNBacyltransferaseBlike-2-(GLYATL2) -0.1044541 -0.0326886
LRRC49 leucine-rich-repeat-containing-49-(LRRC49) -0.113276 -0.0402373
JAK2 Janus-kinase-2-(a-protein-tyrosine-kinase)-(JAK2)-;-see-cat -0.1314697 -0.0525142
ACSF2 Hypothetical1protein1FLJ209201(FLJ20920),1mRNA -0.1976712 -0.05383
ZFYVE19 Zinc1finger1FYVE1domain-containing1protein119 -0.2023717 -0.1007037
C16orf45 chromosome1161open1reading1frame1451(C16orf45) -0.2025902 -0.294768
LRFN4 Leucine-rich1repeat1and1fibronectin1type-III1domain-conta -0.2145687 -0.0165906
ART3 ADP-ribosyltransferase131(ART3) -0.2156815 -0.7288991
BRAF v-raf1murine1sarcoma1viral1oncogene1homolog1B11(BRAF) -0.2327289 -0.0400864
COPZ1 coatomer1protein1complex,1subunit1zeta111(COPZ1) -0.2417656 -0.0503753
SUMO1 SMT31suppressor1of1mif1two131homolog111(yeast)1(SUMO -0.2702305 -0.1349492
WEE1 WEE11homolog1(S.1pombe)1(WEE1) -0.2779083 -0.1411808
TPSD1 Tryptase1delta -0.2844059 -0.130081
GPLD1 glycosylphosphatidylinositol1specific1phospholipase1D11(G -0.3350083 -0.0616363
TPMT thiopurine1S-methyltransferase1(TPMT) -0.3524537 -0.0015808
PLEKHM2 pleckstrin1homology1domain1containing,1family1M1(with1R -0.359433 -0.1164855
C1orf110 Coiled-coil1domain-containing1protein1C1orf110 -0.3731029 -0.2365469
EFCAB4A EF-hand1calcium1binding1domain14A1(EFCAB4A) -0.3905417 -0.4339735
LOC202051 hypothetical1protein1LOC2020511(LOC202051) -0.5320354 -0.6488699
PUS7L Pseudouridylate1synthase171homolog-like1protein -0.7224963 -0.0632583
FIBP fibroblast1growth1factor1(acidic)1intracellular1binding1prot -1.0676641 -0.1202568
DDX43 DEAD1(Asp-Glu-Ala-Asp)1box1polypeptide1431(DDX43) -1.8253505 -0.5233769
MCM10 cDNA1clone1IMAGE:34512141(MCM10) -4.2966154 -3.1014426

 

  • B4) buffer 3 supplemented with compounds known to enhance specific forms of PTM; tested with samples 4, 5, and 10 (non-fasting female, non-fasting male, and fasting female, respectively)

B4 Phosphorylation

Gene Description Samp4 Sample5 Sample10
STK4 Serine/threonine8protein9kinase94 -0.0089982 -0.004821 -0.056209
FLJ22795 hypothetical9protein9FLJ227959(FLJ22795) -2.7402524 -2.0301179 -0.7343827
LMOD1 Leiomodin81 12.8273499 0.41108137 0.10966239
RCOR3 REST9corepressor939(RCOR3) 0.86943111 0.11411907 0.02703298
DNMT3A DNA9(cytosine858)8methyltransferase939alpha9(DNMT3A),9 0.36614796 0.12883865 0.11794506
ISY1 Pre8mRNA8splicing9factor9ISY19homolog 0.34951337 0.10551673 0.01546055
RABL2B RAB,9member9of9RAS9oncogene9family8like92B9(RABL2B) 0.28700246 0.10472062 0.16618374
PIN4 protein9(peptidylprolyl9cis/trans9isomerase)9NIMA8intera 0.20451684 0.11213425 0.24052022
KLRC3 NKG28E9type9II9integral9membrane9protein 0.14389889 0.10606284 0.08339801
DNAL4 dynein,9axonemal,9light9chain949(DNAL4) 0.14221018 0.0297315 0.1346549
SGK3 serum/glucocorticoid9regulated9kinase9family,9member93 0.12704807 0.10331384 0.0161128
CHORDC1 Cysteine9and9histidine8rich9domain8containing9protein91 0.11641043 0.0004399 0.17044143
TRAF1 TNF9receptor8associated9factor919(TRAF1),9mRNA 0.11336669 0.15333571 0.11992884
TPMT thiopurine9S8methyltransferase9(TPMT) 0.10843329 0.07820078 0.07178958
RTP4 receptor9(chemosensory)9transporter9protein949(RTP4) 0.07628673 0.0160817 0.1883564
SPZ1 spermatogenic9leucine9zipper919(SPZ1) 0.07053798 0.0149308 0.2652984
CHML choroideremia8like9(Rab9escort9protein92)9(CHML) 0.06855226 0.16085519 0.06515371
GJB3 gap9junction9protein,9beta93,931kDa9(GJB3),9transcript9var 0.05067757 0.08454953 0.06096041

 

B4 Ubiquitylation

Gene Description Samp4 Sample5 Sample10
KCNG4 potassium8voltage;gated8channel,8subfamily8G,8member8 -0.0016502 -0.0049263 -0.2824588
PPP3R2 protein8phosphatase838(formerly82B),8regulatory8subunit8 -0.0398677 -2.0480832 -0.1140451
ARHGEF1 Rho8guanine8nucleotide8exchange8factor8(GEF)818(ARHGE -0.2981568 -0.2060465 -0.0424863
GNAT2 guanine8nucleotide8binding8protein8(G8protein),8alpha8tra 0.91666007 0.36600867 0.01642788
EXDL2 exonuclease83′;5’8domain;like828(EXDL2) 0.23656465 0.1912862 0.24339316
SUGT1P suppressor8of8G28allele8of8SKP18pseudogene8(S.8cerevisiae 0.16696785 0.21553057 0.11241854
PACSIN2 protein8kinase8C8and8casein8kinase8substrate8in8neurons82 0.08824805 0.04565623 0.08526461
SLC23A2 solute8carrier8family8238(nucleobase8transporters),8memb 0.03901703 0.37332825 0.16014812
ERCC3 excision8repair8cross;complementing8rodent8repair8defici 0.02826044 2.3631335 0.00247819

 

Appendix 2: Ontologies Present within the list of significant (p<0.05) changes.

GO is a unique numeric identifier assigned by the Ontology Consortium; description is the name of that ontology; found is the number of proteins present in the list of changes; annotated is the total number of proteins within that ontology; pVal is the statistical measure for how likely the ontology is present due to chance alone (and p<0.10 is the cutoff of convention among the Consortium); members are the gene abbreviations of the proteins found.

Buffer 1, Phosphorylation 20
Buffer 1, Ubiquitylation 20
Buffer 2, Phosphorylation 20
Buffer 2, Ubiquitylation 21
Buffer 3, Phosphorylation 21
Buffer 3, Ubiquitylation 22
Buffer 4, Phosphorylation 22
Buffer 4, Ubiquitylation 22

 

B1 Phosphorylation

GO Description found annotated pVal members
4693 CDK(activity 2 29 0.0685 cdk9(cdk6
1726 ruffle 2 43 0.0735 cdk6(s100a11
42770 DNA(damage(signal(transduction 2 43 0.0735 abl1(brsk1
31252 leading(edge 2 72 0.0842 cdk6(s100a11
8283 cell(proliferation 5 745 0.0842 stat6(cdk9(il5ra(cdk6(s100a11
7188 GGprotein(signaling 2 84 0.0877 ucn3(gcgr
19933 cAMP(mediated(signaling 2 87 0.0892 ucn3(gcgr
43405 regulation(of(MAPK(activity 2 93 0.0928 trib2(dusp16
9615 response(to(virus 2 98 0.0947 twf2(ifna17

 

B1 Ubiquitylation

GO Description found annotated pVal members
5884 actin)filament 2 25 0.064 tpm4)gas2
42446 hormone)biosynthesis 2 43 0.064 hsd17b8)adm
44255 lipid)metabolism 5 768 0.064 acsl4)mtmr9)pgds)hsd17b8)adm
65002 intracellular)transport)across)a)m 2 69 0.064 acsl4)mtmr9)pgds)hsd17b8)adm
31975 envelope 4 624 0.064 acsl4)atp5c1)nup50)senp2
42445 hormone)metabolism 2 88 0.064 hsd17b8)adm
7507 heart)development 2 94 0.064 mb)adm
51649 establishment)of)cellular)localizat 5 1098 0.064 arnt)cklf)sycn)nup50)senp2
6796 phosphate)metabolism 6 1697 0.0706 oxsr1)pim1)mtmr9)atp5c1)cdc14b)map3k14
46903 secretion 3 384 0.0747 cklf)sycn)adm
5057 receptor)signaling)activity 2 177 0.1 arnt)map3k14

 

B2 Phosphorylation

GO Description found annotated pVal members
22402 cell’cycle’process 7 625 0.0154 cdk5r1′sesn3′dleu1′gas7′pbk’klhdc3′fhit
43005 neuron’projection 3 81 0.0226 cdk5r1′dpysl5′syt1
19209 kinase’activator’activity 2 18 0.0229 cdk5r1′map3k7ip1
51049 regulation’of’transport 3 110 0.0355 cd63′syt1′aaas
43025 cell’soma 2 35 0.0467 cdk5r1′dpysl5
5516 calmodulin’binding 3 141 0.049 edf1′syt1′mylk
30425 dendrite 2 38 0.049 cdk5r1′dpysl5
42995 cell’projection 4 313 0.0494 cdk5r1′parva’dpysl5′syt1
5765 lysosomal’membrane 2 51 0.0543 tmem166′cd63
4672 protein’kinase’activity 7 1158 0.0563 cdk5r1′ulk4′mapk10′pbk’melk’mylk’map3k7ip1
16192 vessicle’mediated’transport 5 606 0.0579 ank1′cd63′rab13′syt1′sh3gl2
7411 axon’guidance 2 56 0.059 cdk5r1′dpysl5
5774 vacuolar’membrane 2 62 0.0648 tmem166′cd63
7178 transmembrane’receptor’serine/t 2 76 0.076 smurf1′map3k7ip1
43065 positive’regulation’of’apoptosis 3 256 0.0816 tia1′cdk5r1′slamf7
7050 cell’cycle’arrest 2 101 0.0928 sesn3′gas7

 

B2 Ubiquitylation

GO Description found annotated pVal members
8283 cell’proliferation 9 745 0.00248 nck1′mxi1′chek1′rapgef3′cd40′alox12′sfn’gtpbp4′kit
44238 primary’metabolism 39 12764 0.00339 nedd8′nck1′tmub1′mrpl43′chek1′rapgef3′rpusd4′suv39h2′cd40
alox12′man2c1′zkscan1′map3k7′vax1′pctk3′apeh’wisp2′g6pd’fkbp2
nagk’smndc1′zkscan3′puf60′ick’melk’glb1l3′mxi1′dffa’stk24′
sds’styx’suclg1′gtpbp4′rpe’mgat4a’ap2m1′znf434′kit’spcs2
45859 regulation’of’kinase’activity 5 237 0.00586 map3k7′sfn’gtpbp4′kit’chek1
5975 carbohydrate’metabolism 8 800 0.00586 man2c1′nagk’mgat4a’sds’rpe’suclg1′glb1l3′g6pd
79 regulation’of’CDK’activity 3 49 0.00618 sfn’gtpbp4′chek1
42127 regulation’of’cell’proliferation 6 469 0.0097 nck1′mxi1′alox12′gtpbp4′kit’chek1
6464 protein’modification 14 2704 0.0106 nedd8′map3k7′pctk3′tmub1′stk24′styx’chek1′rapgef3′spcs2′
mgat4a’melk’ick’gtpbp4′kit
43687 postHtranslational’protein’modific 12 2235 0.0209 nedd8′map3k7′pctk3′stk24′styx’chek1′rapgef3′spcs2′melk’ick’gtpbp4′kit
7243 protein’kinase’cascade 5 376 0.0219 cd40′sectm1′map3k7′ick’kit
45185 maintenance’of’protein’localizatio 2 26 0.0311 mxi1′kdelr1
7249 iKB’NfKb’cascade 3 139 0.0335 cd40′sectm1′map3k7
50870 positive’regulation’of’Tcell’activat 2 41 0.04 map3k7′nck1
46649 lymphocyte’activation 3 155 0.0422 cd40′map3k7′nck1
6915 apoptosis 6 855 0.0453 cd40′smndc1′puf60′alox12′dffa’sfn
46651 lymphocyte’proliferation 2 53 0.0453 cd40′nck1
45321 leukocyte’activation 3 180 0.0453 cd40′map3k7′nck1
30155 regulation’of’cell’adhesion 2 66 0.0573 alox12′gtpbp4
65008 regulation’of’biological’quality 6 953 0.0591 cd40′nck1′alox12′gtpbp4′entpd1′wisp2

 

B1 Ubiquitylation

GO Description found annotated pVal members
5624 membrane+fraction 12 683 3.68E605 kcna4+bid+fibp+kcns3+cyb5a+hsd3b2+pscd2+ighg1+itgb3bp+tspan31+cyp4z1+sptb
42226 interleukin66+biosynthesis 2 12 0.0549 il19+il17f
43687 post6translational+protein+modific 13 2235 0.0649 jak2+fbxo7+braf+art3+wee1+ube2v1+prkg1+cdc7+bub1+pkn3+fbxl12+spsb1+sumo1
8219 cell+death 8 908 0.087 il19+jak2+cfdp1+itgb3bp+bid+braf+il2rb+dbc1

 

B4 Phosphorylation

GO Description found annotated pVal members
8168 methyltransferase/activity 2 271 0.0967 dnmt3a/tpmt
46907 intracellular/transport 3 910 0.0988 dnal4/rtp4/chml
7600 sensory/perception 3 950 0.101 rtp4/chml/gjb3

 

B4 Ubiquitylation

GO Description found annotated pVal members
9416 response+to+light+stimulus 2 81 0.0163 gnat2+ercc3
9314 response+to+radiation 2 101 0.0163 gnat2+ercc3
9628 response+to+abiotic+stimulus 2 165 0.0212 gnat2+ercc3
5886 plasma+membrane 4 3816 0.0672 gnat2+arhgef1+kcng4+slc23a2
51179 localization 4 4481 0.0765 pacsin2+ercc3+kcng4+slc23a2
7600 sensory+perception 2 950 0.0919 gnat2+ercc3
8104 protein+localization 2 961 0.0919 pacsin2+ercc3

 

FAQ
Q1. If positive Mvalues indicate biologically meaningful events (interaction with my POI, or substrates of my POI), do negative Mvalues imply the impossible (e.g., higher interaction with my POI in a sample that did not even contain my POI)?
A1. Negative M-values do indicate that the signal observed at those arrayed proteins was weaker on the case array than on the control. It is (obviously) difficult to conjure any biologically meaningful explanation for the phenomenon, so these signals likely represent noise on the arrays (perhaps there was a scratch on one array, or insoluble material settled on an array at that location etc. etc.). The first thing to consider is that Loess transformation performed in this study is statistically conservative in this regard. It is based on the assumption that true hits should be geographically scattered at random throughout the array, as opposed to showing bias for certain locations. To achieve this effect, it forces values within each of the 48 blocks present on the array to obtain an average signal of zero, and a standard deviation of one. In practice, when there are plenty of good (and positive) signal differences between arrays, it has the effect of exaggerating some of the lesser, or slightly negative differences,
making them appear “more negative” than they in all actuality were. The second consideration is that in any screen of 20,000 questions and a cutoff of p<0.05, we expect to see 1000 changes due to chance alone (by definition). In practice, this number has been shown to be more commonly about 400, indicating good reproducibility of the screening method.

Q2. Does a lower Mvalue indicate a weaker interaction (protein-protein or enzyme-substrate)?

A2. It could certainly be the case that the larger Mvalue represented a stronger interaction, or a biologically real interaction and the small one a spurious interaction. However, it could also mean that both interactions were biologically real, but the large one a structural and stable interaction while the small one represented an enzymatic and transient one (protein-protein) or a biologically critical but rare one (enzyme-substrate). Finally, it is also possible that both interactions were biologically meaningful, stable, and interesting, but the one represented by large Mvalue was for a protein on the array that was easily made recombinantly (expressed at 20mM and purified to 99% homogeneity, for example) while the small one involved a protein that was difficult to make (expressed to 10nM and purified to 50% homogeneity, for example).

Q3. So what do I believe, Mvalue or Pvalue?
A3. Neither one alone, probably. Mvalue captures the “Magnitude” of change between the two arrays, and pValue captures the likelihood that that change (however small or large) was due to chance alone (p<0.05 was used here, which means that there is a 5% or less chance that any of these changes were due to chance alone). If two spots on the case array had values of 1.001 and 1.002, and the corresponding values on the control array were 0.975 and 0.974, a reasonable interpretation would be that two slightly different estimates of the same number (1.0) were reported on both arrays (as captured by the simplified and untransformed Mvalue of 0.027, essentially zero). Given the hyper- reproducible intra-array estimates (stdev of 0.0007 in each case) however, we would see a pValue = 0.027, suggesting something real (less than 3% chance that the change was due to chance alone). On the other hand, if we had values of 100 and 20 on the case array, and values of 3 and 9 on the control, a reasonable interpretation would be that the change was meaningful and real (hypothetical and simplified Mvalue of 54), while the poor reproducibility of intra-array signals on each array (stdev 56.6 and 4.24, respectively) would drive our pValue to 0.43 in this case (a 43% chance that this difference was due to chance alone). The bottom line is that either estimate alone can easily mislead, and both should be used together. In practice, criteria are chosen arbitrarily for each (there is nothing “real” about p<0.05, and a protein with p=0.051 is probably just as “real” as one with p=0.049). In all cases, p<0.05 is utilized per common convention, while Mvalue is individually chosen based on the graphical distribution of hits unique to each project (some proteins simply interact with a LOT more than others, rendering any a-priori assumption difficult to justify). To look at either criterion alone (for example, to capture or “cherry-pick” events like the second hypothetical described above), simply start with the spreadsheet appended with “all” that was provided, and sort by either column (P or M). For purposes of drawing a cutoff of “real” hits, a client may feel free to tighten or loosen the Mvalue chosen by AVMBioMed. Whatever the pValue and Mvalue chosen as cutoff criteria, all hits above that line should be considered equally likely to be real (as borne out by statistical calculation and previous study using independent datasets). The order of hits above cutoff is ALWAYS less important to consider than where that cutoff is drawn.

Q4. This screen failed to identify a positive control protein for my POI (that was indeed present on the
array). What gives?
A4. No screen is perfect -certainly we could have just missed it. Perhaps the pH was slightly off here, or the local concentration of a metabolic cofactor is higher within the cell, etc. Alternatively, we may have missed it given the conservative assumptions with which we analyze the results. Our screens are tailored towards identification of novel interactions with the goal of minimizing false discovery (to maximize your own resources spent on follow-up). As such, we sometimes find that known interactions are not captured. Usually, when we look at such individual proteins, we find data that is indeed consistent with interaction, but simply missed our cutoff criteria for one reason or another (see the 2nd scenario in answer #3 for an example). While initially disappointing, such phenomenon generally increases confidence in the hits that did reach criteria and were reported.

Q5. There is a lot of discussion about “real” hits. You admit that your screen returned some garbage to me?
A5. Yes, we do. There is ultimately no perfect way to order hits or zoom in on EXACTLY which events were biologically real, as is simply the nature of any screen of any size. At p<0.05 in a screen of 20,000 yes/no questions (i.e., “interaction with my POI yes or no?”), we expect to see- by definition of pValue-1000 “yes” answers by chance alone. In practice, the number associated with Snapshot Protemics is usually about 400, indicating high reproducibility for this method. Furthermore, many or most of these are easily identified and removed from further consideration (for example, the slight but reproducible changes resulting in Mvalues of what is essentially zero, as described by the first scenario in answer #3).

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