Chicken Proteomics

 

Original Publication: Sakano H, Zorio DAR, Wang X, Ting YS, Noble WS, MacCoss MJ, Rubel EW, Wang Y (2017). Proteomic analyses of nucleus laminaris identified candidate targets of the fragile X mental retardation protein. J Comp Neurol. 525(15):3341-3359.     PDF

 

Animals          

This study was performed on White Leghorn chick hatchlings (Gallus gallus; post-hatch day 0-4). The first sample type was collected specifically from the NL cell group under a laser micro-dissection microscope (LMD-6000; Leica Microsystems). The second type of tissue sample was collected from the dorsal brainstem at the caudorostral level of NL. Protein preparation was performed, followed by trypsin digestion and peptide purification for mass spectrometry.

Mass spectrometry (MS)

The LTQ-FT Ultra (ThermoFisher Scientific) mass spectrometer was used. Spectra were matched to peptide sequences using SEQUEST. Peptide-spectrum match and peptide identifications were obtained from Percolator (v2.01). Peptides with Percolator with q-value <0.01 were given as input to ID Picker for protein identification. We used a decoy database using scrambled Gallus gallus genome sequence (build 12/17/11). We required at least 2 peptides per protein, each with a q-value (false discovery rate) of <0.01. At least four biological replicates with three technical replicates each were performed. We required each peptide to present in every technical replicate (n=3) and at least 2 peptides per protein for identification.

          Several software programs were used to perform gene ontology analyses of the identified proteins. The first is the DAVID Bioinformatics Resources 6.8 (https://david.ncifcrf.gov/). We used this resource for protein functional annotation and gene ID conversion. Ensembl Bio-mart software (http://www.ensembl.org/biomart/martview/) was also used for gene ID conversion. For identifying transmembrane proteins, we used the TMHMM program version 2.0 located at: http://www.cbs.dtu.dk/services/TMHMM/. Finally, we used the Ingenuity pathway analysis at http://www.ingenuity.com/ as an alternative approach to DAVID for identifying enriched pathways.

 

Download:   Identified Proteins in NL samples         Identified Protein in BS samples (dorsal brainstem)

 

Putative FMRP targets

Comparative analyses with FMRP targets in the mouse brain  download the table of 94 proteins

NL proteomic

 

Thirty-two (32) of the 94 FMRP targets likely to be translated locally     download the table of 32 proteins

 

Cell Growth/Cytoskeleton

CRMP1

collapsin response mediator protein 1

MAP1B

microtubule-associated protein 1B

ACTG1

Actin, gamma 1

SPTBN1

spectrin, beta, non-erythrocytic 1

TUBB

tubulin, beta class I

TUBB2C

tubulin, beta 2C

TUBB2B

tubulin, beta 2B class IIb

TUBB3

tubulin, beta 3 class III

Transport of Ions/Amines (Integral membrane proteins)

ATP6V1B2 (VATB)

ATPase, H+ transporting, lysosomal 56/58kDa, V1 subunit B2

ATP2A2 (SERCA2)

ATPase, Ca++ transporting, cardiac muscle, slow twitch 2

ATP1B1

ATPase, Na+/K+ transporting, beta 1 polypeptide

ATP1A1

ATPase, Na+/K+ transporting, alpha 1 polypeptide

LOC100857345

V-type proton ATPase subunit d 1-like

LOC100858165

V-type proton ATPase subunit d 1-like

Metabolism

HK1

hexokinase 1

ACO2

aconitase 2, mitochondrial

Protein Modification

USP5

ubiquitin specific peptidase 5 (isopeptidase T)

Trafficking of RNA, proteins or vesicles

KIF5C

kinesin family member 5C

DYNC1H1

dynein, cytoplasmic 1, heavy chain 1

DNM1

dynamin 1

ARF1

ADP-ribosylation factor 1

AMPH

amphiphysin

SNAP25

synaptosomal-associated protein, 25kDa

SYNJ1

synaptojanin 1

NSF

N-ethylmaleimide-sensitive factor

SNAP91

synaptosomal-associated protein, 91kDa homolog (mouse)

Signaling

CALM

calmodulin 2 (phosphorylase kinase, delta)

MBP

myelin basic protein

NDRG4

N-myc downstream regulated gene family member 4

Translation

EEF2

eukaryotic translation elongation factor 2

EEF1A1

eukaryotic translation elongation factor 1 alpha 1

EEF1A2

eukaryotic translation elongation factor 1 alpha 2