Lec 9 Label free ms/seldi TOF Flashcards
what level is label free proteomics quantification and identification carried out at
peptide level
what level is Seldi-TOF quantification carried out at
protein level quantification but identification of the protein is difficult
what does Seldi-TOF stand for
surface-enhanced laser desorption/ionization Time of Flight
what are the fundamental steps of all label free quantitative proteomics methods
1 - sample preparation, 2 - sample separation, 3 - data analysis
what is involved in fundamental step 1 of label free quantification
sample preparation: protein extraction, reduction, alkylation, and digestion
what is involved in fundamental step 2 of label free quantification
sample separation: by liquid chromatography (LC or LC/LC) and analysis by MS/MS
what is involved in fundamental step 3 of label free quantification
data analysis: peptide/protein identification, quantification, and statistical analysis
advantages of label free proteomics
straightforward and inexpensive, unmodified peptides/proteins, minimal manipulation of the sample, can be used on any type of biological material
what are the strategies for label free quantification
1 - spectral counts, direct MS-signal comparison
what is spectral counting
counting the number of MS/MS spectra identifying a given peptide/protein which is a simple approach for relative quantification (without requiring stable isotope labelling)
what is the rationale behind spectral counting
the frequency for which a peptide is selected for MS/MS fragmentation is positively correlated to its quantity - the greater the amount of peptide the longer it takes to elute from the column meaning more time for MS to detect it
what is ion intensity (another method of label free quantification)
following accurate alignment of detected LS-MS features across different analyses the MS peak intensities can be compared among different samples for relative quantification
protein results of ion intensity
quantitative LC-MS data and qualitative (peptide identification) LC-MS results are brought together at the protein level, showing expression changes and ANOVA p-values between experimental groups
challenges of ion intensity label free
-highly reproducible LC-MS analysis
-retention time shift
-fluctuations in MS signal intensity
-peptide identification in separated MS/MS
-complex samples
-overlapping signals
-misaligned peptides
-large sample size
-column degradation
