Mass Spectrometry

Our coverage of mass spectrometry will begin with the basic elements of a mass spectrometer and then focus on some of the more recent developments that enable these approaches to be so successful with biomacromolecules.  Refer to the web sites for further information.

Overview:  - There are many web site tutorials on Mass Spec; Refer to this WORD document below, or one of many  tutorials on the ASMS (American Society for Mass Spectrometry) web site. 

I.  General Principles: (Most of the following is covered in the second web reference above (AMAS) - and summarized with text and figures in this MS_document and these PPT slides and notes)

What does a mass spectrometer do?

A mass spectrometer produces charged particles (ions) from the chemical substances that are to be analyzed. The mass spectrometer then uses electric and magnetic fields to measure the mass ("weight") or mass / charge ratio of the charged particles.

 

II.  Basic Elements of a Mass Spectrometer: 

How does a mass spectrometer work?

There are many different kinds of mass spectrometers, but all use magnetic and/or electric fields to exert forces on the charged particles produced from the chemicals to be analyzed. A basic mass spectrometer consists of three parts:

  1. A source in which ions are produced from the chemical substances to be analyzed.

    Source:  Forming Charged Particles (Ions):

  2. An analyzer in which ions are separated according to mass.

    Analyzer

    System Highlights

    Quadrupole

    Unit mass resolution, fast scan, low cost

    Sector (Magnetic and/or Electrostatic)

    High resolution, exact mass

    Time-of-Flight (TOF)

    Theoretically, no limitation for m/z maximum, high throughput

    Ion Cyclotron Resonance (ICR)

    Very high resolution, exact mass, perform ion chemistry

  3. A detector which produces a signal from the separated ions.

Linked Systems:
 

GC/MS:

Gas chromatography coupled to mass spectrometry

LC/MS:

Liquid chromatography coupled to electrospray ionization mass spectrometry

MS/MS

Tandem mass spectrometry

III. Focus on Recent Advances for Use with Biomacromolecules  

Application to Biological Macromolecules (Macromolecular ions):

For many years people recognized the value of mass spectrometry but faced the difficulty of vaporizing a macromolecule in a vacuum without unacceptable degradation.  Recent developments have made mass spectrometry a major tool for the analysis of a variety of biomacromolecules.

A) Advances in Ion Sources:

1) Electrospray Ionization (ESI):

The advantage of this method is that it is nondestructive so it is well suited for macromolecules.  A tiny, charged microdroplets containing the macromolecule in solution are passed through a fine needle and sprayed into the mass spectrometer in an electric field – the solvent evaporates leaving the macroion in the vacuum for analysis.  This technique typically produces a wide range of ions and often other ions like K+ or other macromolecules tend to associate with the given macromolecule, so the mass spectrum is more complicated but can also provide a wealth of information. 

Because electrospray ionization methods normally puts many charges (z) on the macroion in the form of protons, the m/z ratio for this method is still rather modest with m/z ~ 2000-4000 range.  Even quadrupole mass spectrometers with limited mass ranges (~800 – 4000) can be used with this method. 

2) Matrix-Assisted Laser Desorption-Ionization (MALDI): 

Laser desorption methods use a pulsed laser to desorb species from a target surface.  A mass analyzer such as time-of-flight (TOF) is used that is compatible with pulsed ionization methods. 

Direct laser desorption relies on the very rapid heating of the sample or sample substrate to vaporize molecules so quickly that they do not have time to decompose. This is good for low to medium-molecular weight compounds and surface analysis. The more recent development of matrix-assisted laser desorption ionization (MALDI) relies on the absorption of laser energy by a matrix compound. MALDI has become extremely popular as a method for the rapid determination of high-molecular-weight compounds.  Macromolecules are implanted into a solid chemical matrix, usually an aromatic compound like 2,5-dihydroxybenzoic acid that strongly absorbs UV light.  The analyte is dissolved in a solution containing an excess of a matrix such as sinapinic acid or dihydroxybenzoic acid that has a chromophore that absorbs at the laser wavelength.  A small amount of this solution is placed on the laser target. The matrix absorbs the energy from the laser pulse and produces a plasma that results in vaporization and ionization of the analyte macromolecule. 

MALDI requires a mass analyzer that is compatible with pulsed ionization techniques (TOF). Accuracy to better than 0.1% can be achieved and the mass spectrum is usually very clean with the “molecular” ion plus relatively few multiples.  Protein molecules of mass greater than 150,000 Da have analyzed with accuracy using MALDI-TOF.

3) Capillary Electrophoresis (CE) and ESI:

Capillary electrophoresis is similar to gel electrophoresis but without the gel.  Very small quartz capillaries (~50-100 mm in diameter x 20-60 cm in length) are used.  The high electrical resistance of the small capillaries allows very high voltages to be applied without large current leading to rapid separation (minutes) with little convection mixing.

Another major advantage of this technique is the use of very small samples – mass sensitivity in the femtomole (10-15) and even zeptomole (10-21) ranges.   Coupling CE with electrospray ionization (ESI) mass spectrometry has proven practical for the analysis of tiny amounts of biomacromolecules.  

B) Advances in Mass Analyzers:

1) Time-of Flight (TOF) Mass Spectrometer: 

Generate positive ions in a short source, accelerate all ions to same kinetic energy KE = (Ze)Es  where”Ze” is the charge, “E” the electric field, and “s” the length of the source region, allow ions to emerge into a “field-free, drift” region of length D.  Since KE is also = ½(mv2), heavier ions will move more slowly than light ions as they “drift” to the detector.  If t is the time for an ion to traverse the drift region, the mass/charge ratio can be given by:

                                    (m/Z) = 2(Ze)s(t/D)2 , or  m = [2eEs(1/D)2] Z t2

                                                mass = (constant) x Z x t2   

2) Quadrupole: 

A quadrupole mass filter consists of four parallel metal rods (Figure). Two opposite rods have an applied potential of (U+Vcos(wt)) and the other two rods have a potential of -(U+Vcos(wt)), where U is a dc voltage and Vcos(wt) is an ac voltage. The applied voltages affect the trajectory of ions traveling down the flight path centered between the four rods. For given dc and ac voltages, only ions of a certain mass-to-charge ratio pass through the quadrupole filter and all other ions are thrown out of their original path. A mass spectrum is obtained by monitoring the ions passing through the quadrupole filter as the voltages on the rods are varied.  

IV.  Sample Applications 

Sequence Analysis Using Mass Spectrometry:  Since the masses of peptides and protein fragments can be measured with great accuracy, it is possible to determine amino acid content from mass or the sequence of a protein fragment if the whole sequence is known.