How Does Mass Spectrometry Identify Proteins?

fingerprint

Protein Identification

Mass spectrometers identify proteins in a manner analogous to using fingerprints to identify a person. Each protein has a mass spectrum with unique characteristics. To minimize confusion, proteins must first be separated from protein mixtures; then they can be examined with a mass spectrometer.

The most common form of protein identification with mass spectrometry uses a tandem mass spectrometer (abbreviated MS/MS). As explained below, identifying a protein with MS/MS is like identifying a person using fingerprints.

Clarification: In the proteomics field, the term "Protein Mass Fingerprinting", PMF, describes a simpler procedure using a simpler mass spectrometer. The broader analogy of MS/MS to fingerprinting is used here only to explain the concepts.

Fingerprint Identification

Fingerprints at a crime scene might be mixed in with hundreds of other fingerprints. The first step is to collect and separate the fingerprints. The common way to separate fingerprints is to look at different surfaces. If they overlap, fluorescence can sometimes separate them.

Protein Identification

A biological sample contains hundreds or thousands of proteins, all mixed together. The first step is to separate them. One common technique uses two-dimensional gels. The spots on the gel are different proteins, separated horizontally by pH and vertically by mass (the two dimensions).

Fingerprints are identified by looking at each finger — that is, the hand print at the top of the page is considered as the four individual prints below. A protein's peptides are analogous to these individual fingers.

While each person has only 4 fingers, one protein may have 18 peptides, another 27 peptides and yet another only 6 peptides.

VHLTPEEK
SAVTALWGK
VNVDEVGGEALGR
LLVVYPWTQR
FFESFGDLSTPDAVMGNPK
VK
AHGK
K
VLGAFSDGLAHLDNLK
GTFATLSELHCDK
LHVDPENFR
LLGNVLVCVLAHHFGK
EFTPPVQAAYQK
VVAGVANALAHK

Mass spectrometers can identify proteins better if they are first broken down into tryptic peptides. Tryptic peptides are chains of amino acids that occur when the proteins are digested with the enzyme trypsin.

The tryptic peptides for hemoglobin are shown here.

The fingerprints have to be further sorted out by whether they have a whorl, a loop, or arch before they can be identified.

The peptides have to be further sorted before they can be identified. This is often done in the following two steps:

  1. HPLC can separate peptides based on how soluble the peptides are — that is, the peptide hydrophobicity.
  2. Mass spectrometry separates the peptides based upon their mass. This is the first mass spectrometer of the two in a tandem mass spectrometer instrument.

Now the individual fingerprints are examined in detail. Certain identifying features are located, such as a core (the center of a loop) and a delta (a place where ridges divide).

Now the second part of the tandem mass spectrometer examines the individual peptides in detail for identifying features — the distances between the peaks in the mass spectral graph. The peaks represent the number of ions in the peptide's spectrum that have a certain mass and charge.

A person is identified by matching the identifying features of the fingerprints to a database of fingerprints. An identification is more believable if it is based on matching fingerprints from several fingers.

A protein is identified by matching the identifying features of the peptides to a database of proteins. An identification is more believable if it is based on matching mass spectra from several peptides.

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