Primary structural documentation of the major urinary protein of the Indian commensal rat (Rattus rattus) using a proteomic platform.
OBJECTIVE
A number of proteome studies have been performed recently to identify pheromone-related protein expression and their molecular function using genetically modified rodents' urine. However, no such studies have used Indian commensal rodents; interestingly, in a previous investigation, we confirmed the presence of volatile molecules in commensal rodents urine and these molecules seem to be actively involved in pheromonal communication. Therefore, the present study aims to identify the major urinary protein [MUP] present in commensal rat urine, which will help us to understand the protein's expression pattern and intrinsic properties among the rodents globally.


METHODS
Initially, the total urinary proteins were separated by 1-D and 2-D electrophoresis and then subsequently analyzed by Matrix Assisted Laser Desorption Ionization-Time of Flight and Mass Spectrometer (MALDI-TOF/MS). Furthermore, they were then fragmented with the aid of a Tandem Mass Spectrometer (TOF/TOF) and the identified sequences aligned and confirmed using similarity with the deduced primary structures of members of the lipocalin superfamily.


RESULTS
The SDS-PAGE protein profiles showed distinct proteins with molecular masses of 15, 22.4, 25, 28, 42, 50, 55, 68, and 91 kDa. Of these proteins, the 22.4 kDa protein was considered to be target candidate. When 2D elctrophoresis was carried out, about approximately 50 spots were detected with different masses and various pI ranges. The 22.4 kDa protein was found to have a pI of about 4.9.This 22.4 kDa protein spot was digested and subjected to mass spectrometry; it was identified as rat MUP. The fragmented peptides from the rat MUP at 935, 1026, 1192, and 1303 m/z were further fragmented with the aid of MS/MS and generated de novo sequence and this confirmed this protein to be the MUP present in the urine of commensal rats.


CONCLUSIONS
The present investigation confirms the presence of MUP with a molecular mass of 22.4 kDa in the urine of commensal rats. This protein may be involved in the binding of volatile molecules and opens up a discussion about how volatile and non-volatile molecules in the commensal rats' urine may contribute chemo-communication.
Rajkumar
Ramalingam
R
Center for Pheromone Technology, Department of Animal Science, Bharathidasan University, Tiruchirappalli-620 024, India.
Prakash
Subramaniya
S
Archunan
Govindaraju
G
Sowdhamini
Ramanathan
R
D000595:Amino Acid Sequence; D000818:Animals; D017124:Conserved Sequence; D015180:Electrophoresis, Gel, Two-Dimensional; D007194:India; D054834:Lipocalins / Q000737:chemistry; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*; D051381:Rats; D016415:Sequence Alignment / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry / Q000379:methods*
D054834:Lipocalins; D011506:Proteins; C035737:major urinary proteins