Proteomics

Dataset Information

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Monitor precursor mass shifts of Halobacterium salinarum


ABSTRACT: Approximately 2x106 CL1-5 and H23 cells were seeded on 10-cm dishes with RPMI-1640 medium containing 10% fetal bovine serum. After the cells covered 80% of the dish, cells were serum-starved for 24 h in RPMI-1640 medium. CL1-5 and H23 cell lysates were harvested, trypsinized and analyzed by using LTQ-Orbitrap XL and LTQ-Orbitrap Discovery, respectively. Halobacterium salinarum NRC-1 (ATCC700922) starter culture was prepared by inoculating a single colony in 5 mL of Halobacterium medium containing trace metals (CM+) and grown at 37 °C with shaking at 225 rpm for one week. An aliquot of 300 μL of the culture was spread on 15-cm diameter CM+ plates containing 2% of agarose. After 7~10 days of incubation at 37 °C, the GVs from cells on 10 plates were harvested using the centrifugation facilitated flotation method as previously described except the GVs were washed for a total of 5 times instead of the suggested 3 times to remove nonspecific bound proteins.(29) After collecting the gas vesicles in the primary cell lysate, the cell membrane and residual GV were removed by ultracentrifugation at 53 000× g, 4 °C for 16 h twice to obtain the GV-depleted lysate (GVD). GVD lysate was harvested, trypsinized and analyzed by using LC-MS/MS ( LTQ-Orbitrap XL). The H. salinarum strain NRC-1 was grown in complex medium (CM+) at 37 oC with shaking (200 rpm) to an OD600 of 0.5 (early exponential phase. The cultures were then subjected to an additional hour of incubation at 37 oC for preparation of proteins. The total lysate was harvested, trypsinized and analyzed by using LC-MS/MS (LTQ-Orbitrap Discovery). All MS raw data in Thermo XCalibur (version 2.0.7) binary format were converted to the mzXML open data format using a modified version of the ReAdW program. SEQUEST (version 27) was applied to search the MS/MS spectra against the forward and reverse (decoy) protein sequences of H. salinarum (2,427 proteins; EMBL-EBI Integr8, www.ebi.ac.uk/integr8/EBI-Integr8-HomePage.do) or human (38,425 proteins) plus bovine (22,863 proteins) (GenBank reference sequences, www.ncbi.nlm.nih.gov). SEQUEST results were further processed using the Trans Proteomic Pipeline.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Homo Sapiens (human) Halobacterium Salinarum

SUBMITTER: Lichieh Julie Chu  

LAB HEAD: Wailap Victor Ng

PROVIDER: PXD000082 | Pride | 2020-01-14

REPOSITORIES: Pride

Dataset's files

Source:
Action DRS
CL1_5_lysate.pep.xml Pepxml
CL1_5_lysate.prot.xml Xml
CL1_5_lysate_400_2000.mzXML Mzxml
CL1_5_lysate_400_2000_081015042515.mzXML Mzxml
CL1_5_lysate_400_521.mzXML Mzxml
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Publications

Large precursor tolerance database search - a simple approach for estimation of the amount of spectra with precursor mass shifts in proteomic data.

Weng Rueyhung Roc RR   Chu Lichieh Julie LJ   Shu Hung-Wei HW   Wu Timothy H TH   Chen Mengchieh Claire MC   Chang Yuwei Y   Tsai Yihsuan Shannon YS   Wilson Michael C MC   Tsay Yeou-Guang YG   Goodlett David R DR   Ng Wailap Victor WV  

Journal of proteomics 20130808


Mass measurement and precursor mass assignment are independent processes in proteomic data acquisition. Due to misassignments to C-13 peak, or for other reasons, extensive precursor mass shifts (i.e., deviations of the measured from calculated precursor neutral masses) in LC-MS/MS data obtained with the high-accuracy LTQ-Orbitrap mass spectrometers have been reported in previous studies. Although computational methods for post-acquisition reassignment to monoisotopic mass have been developed to  ...[more]

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