Lecture 5 Flashcards
(16 cards)
What is the primary purpose of mass spectrometry?
To identify unknown compounds by determining the weight of their various ions.
How does a mass spectrometer differentiate between heavier and lighter molecules?
Heavier ionized molecules are harder to move/deflect less by a given force than lighter ones.
List the three basic components of a mass spectrometer.
Ionization Chamber, Mass Analyzer, and Detector.
What is the function of the Ionization Chamber?
Converts sample molecules into charged ions.
What is the function of the Mass Analyzer?
Converts sample molecules into charged ions.
What is the function of the Detector?
Measures the abundance of each separated ion, creating a mass spectrum.
What information is presented on a mass spectrum’s X and Y axes?
X-axis: Mass-to-charge ratio (m/z); Y-axis: Relative abundance/intensity of ions.
What is the classical method for protein sequencing, and what is its main limitation?
Edman degradation. Limitation: generally limited to sequencing peptides up to 50-60 amino acids long, and requires relatively large amounts of pure protein.
Briefly explain the principle of Edman degradation.
Sequentially removes one amino acid at a time from the N-terminus of a peptide, which is then identified.
How has mass spectrometry changed protein sequencing?
Made it faster, more sensitive, and able to handle complex mixtures, largely replacing traditional methods.
What is Tandem Mass Spectrometry (MS/MS) used for in protein sequencing?
To sequence peptides. A specific peptide ion is selected (MS1), fragmented, and the fragment masses are measured (MS2) to determine its amino acid sequence.
How are disulfide bonds handled before protein sequencing by MS/MS?
They must be broken (reduced) to obtain linear peptides for accurate fragmentation. This is often done using reagents like beta-mercaptoethanol, and then the thiols are protected to prevent re-oxidation.
Why are large proteins often digested into smaller peptides for MS/MS sequencing?
Large proteins are too complex to sequence directly. Smaller peptides allow for more efficient and accurate mass analysis and sequencing.
What is the role of proteases in protein sequencing workflows?
Proteases (like trypsin) are enzymes that specifically cut proteins into smaller, overlapping peptide fragments suitable for mass spectrometry.
What is the advantage of using Liquid Chromatography coupled with Mass Spectrometry (LC-MS/MS)?
LC-MS/MS separates complex peptide mixtures before mass spectrometry, improving sensitivity and coverage for protein identification in complex samples.
Briefly explain protein identification using mass spectrometry data and database searching.
Experimental peptide mass fingerprints and MS/MS fragmentation patterns are compared against theoretical patterns from protein/DNA sequence databases to identify the protein.
