scholarly journals Evaluation of Normalization Methods on GeLC-MS/MS Label-Free Spectral Counting Data to Correct for Variation during Proteomic Workflows

2011 ◽  
Vol 22 (12) ◽  
pp. 2199-2208 ◽  
Author(s):  
Emine Gokce ◽  
Christopher M. Shuford ◽  
William L. Franck ◽  
Ralph A. Dean ◽  
David C. Muddiman
Keyword(s):  
Label Free ◽  
Spectral Counting ◽  
Counting Data ◽  
Normalization Methods

scholarly journals Development and Evaluation of Normalization Methods for Label-free Relative Quantification of Endogenous Peptides

2009 ◽  
Vol 8 (10) ◽  
pp. 2285-2295 ◽  
Author(s):  
Kim Kultima ◽  
Anna Nilsson ◽  
Birger Scholz ◽  
Uwe L. Rossbach ◽  
Maria Fälth ◽  
...  
Keyword(s):  
Relative Quantification ◽  
Label Free ◽  
Endogenous Peptides ◽  
Normalization Methods ◽  
Free Relative

scholarly journals Improved LC-MS chromatographic alignment increases the accuracy of label-free quantitative proteomics: Comparison of spectral counting versus ion intensity-based proteomic quantification strategies

2017 ◽  
Author(s):  
Daniel H.J. Ng ◽  
Jonathan D. Humphries ◽  
Julian N. Selley ◽  
Stacey Warwood ◽  
David Knight ◽  
...  
Keyword(s):  
Cell Biology ◽  
Low Cost ◽  
Quantitative Description ◽  
Protein Quantification ◽  
Global Changes ◽  
Label Free ◽  
Spectral Counting ◽  
Differential Abundance ◽  
Mass Spectrometers ◽  
Wide Range

AbstractThe ability to provide an unbiased qualitative and quantitative description of the global changes to proteins in a cell or an organism would permit the systems-wide study of complex biological systems. Label-free quantitative shotgun proteomic strategies (including LC-MS ion intensity quantification and spectral counting) are attractive because of their relatively low cost, ease of implementation, and the lack of multiplexing restrictions when comparing multiple samples. Owing to improvements in the resolution and sensitivity of mass spectrometers, and the availability of analytical software packages, protein quantification by LC-MS ion intensity has increased in popularity. Here, we have addressed the importance of chromatographic alignment on protein quantification, and then assessed how spectral counting compares to ion intensity-based proteomic quantification. Using a spiked-in protein strategy, we analysed two situations that commonly arise in the application of proteomics to cell biology: (i) samples with a small number of proteins of differential abundance in a larger non-changing background, and (ii) samples with a larger number of proteins of differential abundance. To perform these assessments on biologically relevant samples, we used isolated integrin adhesion complexes (IACs). Technical replicate analysis of isolated IACs resulted in a range of alignment scores using the Progenesis QI software package and demonstrated that higher LC-MS chromatographic alignment scores increased the precision of protein quantification. Furthermore, implementation of a simple sample batch-running strategy enabled good chromatographic alignment for hundreds of samples over multiple batches. Finally, we applied the sample batch-running strategy and compared quantification by LC-MS ion intensity to spectral counting and found that quantification by LC-MS ion intensity was more accurate and precise. In summary, these results demonstrate that chromatographic alignment is important for precise and accurate protein quantification based on LC-MS ion intensity and accordingly we present a simple sample re-ordering strategy to facilitate improved alignment. These findings are not only relevant to label-free quantification using Progenesis QI but may be useful to the wide range of MS-based quantification strategies that rely on chromatographic alignment.

scholarly journals A label-free quantification method by MS/MS TIC compared to SILAC and spectral counting in a proteomics screen

PROTEOMICS ◽  
2008 ◽  
Vol 8 (5) ◽  
pp. 994-999 ◽  
Author(s):  
John M. Asara ◽  
Heather R. Christofk ◽  
Lisa M. Freimark ◽  
Lewis C. Cantley
Keyword(s):  
Label Free ◽  
Spectral Counting ◽  
Quantification Method ◽  
Label Free Quantification ◽  
Free Quantification

69 DIFFERENTIAL AND QUANTITATIVE ANALYSIS OF DOG OVIDUCTAL FLUID

2012 ◽  
Vol 24 (1) ◽  
pp. 147 ◽  
Author(s):  
K. Reynaud ◽  
V. Labas ◽  
G. Harichaux ◽  
S. Thoumire ◽  
M. Z. Tahir ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Protein Identification ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Limiting Factors ◽  
Label Free ◽  
Spectral Counting ◽  
Sds Page ◽  
Oviductal Fluid

The major reproductive peculiarity of the bitch is that ovulation releases prophase I (germinal vesicle, GV, immature) oocytes. Resumption of meiotic maturation, as well as fertilisation and embryonic development to the morula stage occur in the oviduct. Because the dog is a biomedical model for human diseases and also a model for endangered canid species, the development of assisted reproduction techniques would be of great interest. To date, in vitro-produced canine embryos remain exceptional and no puppy has been born. The main limiting factors of in vitro embryo production are the low oocyte maturation rates, the poor oocyte quality and the high polyspermy. A better knowledge of the composition of oviductal fluid during the periovulatory period may help to mimic the in vivo conditions for in vitro oocyte culture and, thereafter, their fertilisation and embryonic development. The objective of this study was to analyse the oviductal fluid by a label-free quantitative proteomic workflow based on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) protein separation, nano-scale liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) analysis and quantitative method using spectral counting. Ovarian cycles were followed by vaginal smears, ultrasonography and progesterone blood assays. Oviductal fluids were collected from 3 beagle bitches, after ovariectomies performed 3.5 days after ovulation. After dissection, the ampulla and isthmus were separated and flushed with 50 μL of PBS. Oviductal fluids were submitted to 1D SDS-PAGE and all bands were digested with trypsin. Peptide extracts were analysed on an Ettan multidimensional LC (MDLC) system coupled to a linear ion trap quadrupole (LTQ) mass spectrometer. After protein identification using Mascot server and with Swiss-Prot and National Center for Biotechnology Information (NCBI) databases, bioinformatic processing of data and statistic analysis (t-test with P < 0.05) were performed using the spectral counting quantitative module of the Scaffold software. Using this strategy, 427 proteins were qualitatively identified in canine oviductal fluid. Three proteins were specific of the ampulla, 10 specific of the isthmus and 414 were found in both oviductal parts. Among these common proteins, some were differentially expressed, from 1.25 to 9 times higher (HV303_Human, RLA2_Horse, SPRL1_Human, SODC_CANFA, PROF1_Human, ARF4_Bovin and TRXR1_Bovin). The gene ontology analysis displayed biological pathways specific to the biology of reproduction (6 proteins; RUVB1_Human, OVGP1_Pig, STAT3_Human, PLAK_Human, GPX3_Rat and DYL1_Human). These candidate proteins and especially oviduct-specific glycoprotein and glutathione peroxidase, will now be validated by immunodetection methods.

scholarly journals A systematic evaluation of normalization methods in quantitative label-free proteomics

2016 ◽  
pp. bbw095 ◽  
Author(s):  
Tommi Välikangas ◽  
Tomi Suomi ◽  
Laura L. Elo
Keyword(s):  
Systematic Evaluation ◽  
Label Free ◽  
Normalization Methods
Sign in / Sign up

Export Citation Format

Share Document