Mass Spectrometry basics and terminology
Identify the three major steps of Mass Spectrometry
what are:
1. Produce ions from your analyte/sample (e.g ESI)
2. Manipulate these ions and organize them based on their mass-to-charge ratios using magnetic fields or an electric current. These can be adjusted to change how they move based on their mass-to-charge ratios
3. The detector in the Mass Spectrometer produces a spectrum of these ions in the form of peaks which can be analyzed to determine the mass of molecules such as proteins or amino acids.
What is ESI?
ESI is a method for creating ions of an analyte. A potential difference is created between a capillary tube and the direction the ions are traveling (entry point to a detector). The capillary tube is filled with a solution containing an analyte. The solution is sprayed out of the tube, and ions are created and dragged towards the negative side of the machine.
This piece of metal is an early attempt at a ion detector. It works by having ions hit the plate, generating a current which can be analyzed to determine how many charges are hitting the plate. This device is considered to be unreliable
What is: a Faraday Cup.
Glu Fib positive spectrum. I am holding it in my hand because this site is charging me 20 dollars to upload images!
1571-Glu-fib with an additional proton so this corresponds to an m/z value of 1571. Neutral mass is 1570 Da.
786 corresponds to glu-fib with two protons. So the mass is 1572 but its m/z=786
Calculate the mass from the 786 peak
786*2=1572 This is the mass of glufib plus two protons
1572-2H=1570 This is the neutral mass of glu fib
Define m/z, and why it is useful.
m/z is the mass-to-charge ratio of ions detected by a mass spectrometer. These values are usually listed above individual peaks in a mass spectrum So, say you have a protein with a mass of 1000 Daltons. Contained in that mass are 2 protons (a 2+ charge). You would divide 1000 Daltons by 2 to get 500, which is the m/z value for that specific ion. m/z values are useful because you can use them to calculate the neutral mass of a peptide or protein of interest.
What types of molecules are most often analyzed using ESI?
What are: small molecules, peptides, proteins and carbohydrates.
This detector is, like the one above, just a detector and not a mass analyzer. It works by having the ions bounce off of its surfaces, multiplying the charges and effectively creating charges to be read by an analyzer. This creates a stronger signal, which can be more easily analyzed.
What is: A dynode
Glu-Fib negative spectrum
1569-mass of deprotonated glu-fib so 1569 is its m/z value
784-is the m/z of glufib after it loses 2 protons.
Calculate the mass at 784 peak
784*2=1568 This is the mass of glufib minus 2 protons
1568+2H=1570 This is the mass of neutral glufib
Monoisotopic mass vs Average mass. Explain the difference, and when to or when not to use each when reporting data.
Monoisotopic mass is the exact mass of the lightest stable isotope of an element. Average mass is the average of all isotopes of a single element, with mass contributions being dependent on the abundance of a specific isotope in nature.
When analyzing your mass spectra, the monoisotopic peak will appear on the left side of your spectrum if you are analyzing a lighter amino acid or protein. If your protein is much heavier, the peak may not appear as much heavier naturally occurring isotopes are more prevalent, rendering the monoisotopic peak very small and impossible to report a value for it.
Monoisotopic-more precise
This MASS ANALYZER uses DC and AC currents. These currents can be set at specific voltages to allow for only ions with specific m/z values to be analyzed.
What is: A Quadrupole Mass Ion Filter
Electrospray Ionization spectrum of Apo-myoglobin
Describe the spectrum on camera
List some basic terminology of amino acids, and explain how MS relates to amino acids
Amino acids are building blocks of proteins. The N-terminus is the end containing a free amine group (NH2), and the C-terminus is the end containing a free carboxylic acid (COOH).
When analyzing a peptide or a protein (sequence of amino acids) using MS, we can use the m/z values of ions (individual amino acids) to determine a neutral mass, which can then be compared to a table of amino acids to sequence said peptide/protein.
What is the purpose of Sheath gas? What about Sweep gas?
Sheath gas (N2 gas) is used to help direct ions towards the mass spectrometer. It keeps the stream of ions narrower. This gas is also heated, which helps to evaporate the solvent from the ions.
Sweep gas is also heated N2 gas but is positioned at the opposite end to the sheath gas. This gas helps to further evaporate excess solvent. It also keeps any neutral particles (things we don't want to be detected) out of the ion detector.
This Mass Analyzer includes a region in which ions travel through at different speeds. Meaning ions with larger masses will travel through it more slowly. This allows for specific separation of ions based on mass
What is: A Time of Flight Mass Analyzer
Spectrum for unknown protein
Which type of mass spectrometry was used in lab this quarter to determine the mass of an unknown protein
What is: Ion Trap Mass Spectrometry with an Electrospray Ionization source.
What is unique about ion formation in an ESI mechanism
There are two proposed mechanisms for ion formation in ESI! Scientists are at this point unsure as to how these ions are actually formed.
Are ions ejected out of the solvent droplets?
Does the solvent evaporate away, leaving the ions behind in the gas phase?
This Mass Analyzer contains a quadrupole; however, instead of ejecting ions out, it traps the ions inside, allowing for analysis. DC voltage is used to create a potential well in order to trap the ions.
What is: A quadrupole Ion trap.