Mass spectrometry (MS) is an analytical method used to measure ions based on their mass-to-charge ratio, written as m/z.
For peptide research, this information can help researchers compare an observed molecular mass with the mass expected for a specified peptide. When tandem mass spectrometry (MS/MS) is used, fragment-ion data can provide additional evidence relevant to a proposed peptide identification.
When mass spectrometry is combined with liquid chromatography, the method is called LC-MS. Liquid chromatography separates the components of a sample, while mass spectrometry provides information about the ions detected during that separation. Using the two methods together can provide complementary information about a peptide sample.
What Mass Spectrometry Measures
A mass spectrometer does not simply weigh a peptide. Instead, it finds charged particles called ions and measures their mass-to-charge ratio, or m/z.
A peptide can carry more than one electrical charge. As a result, the same peptide can produce signals at different m/z values. Researchers can use m/z measurements and an assigned charge state to calculate an estimated molecular mass.
This is why a mass spectrometry report may list an m/z value rather than simply giving the molecular weight of a peptide.
Molecular Mass and Peptide Identity
Researchers can compare an observed molecular mass with the expected mass of a peptide. If the two values are consistent, the result can support the expected molecular assignment.
However, matching molecular mass alone does not necessarily provide enough information to confirm the entire peptide sequence. MS/MS can provide additional information by breaking peptide ions into smaller pieces. The resulting fragments create a pattern that can be used to investigate the peptide’s sequence.
This means that molecular mass and peptide identity are related, but they aren’t exactly the same thing. A mass match provides one piece of information, while MS/MS data can provide additional evidence.
How LC-MS Complements Chromatography
LC-MS combines two analytical methods that provide different types of information.
Liquid chromatography separates the components in a sample. The resulting chromatogram can show different peaks representing components detected during the analysis.
Mass spectrometry provides information about the ions associated with those components. This can help researchers investigate the molecular mass of material associated with a particular chromatographic peak.
Together, chromatography and mass spectrometry can provide complementary information about separated components and the ions associated with them. When the method is appropriate for the sample and purpose, LC-MS/MS data can support a proposed peptide identity assignment.
What a Mass Spectrum Shows
A mass spectrum is displayed as a series of peaks. The position of each peak represents an m/z value, while the height of the peak represents its relative signal under the conditions used for the analysis. Peak height alone does not establish the amount or purity of a component.
During MS/MS, peptide ions are broken into smaller fragments. These fragments produce additional peaks. The pattern of those peaks can provide information about the peptide from which they came.
Comparing Observed and Expected Mass
Researchers can calculate an expected molecular mass from a peptide’s known sequence or composition. The measured mass can then be compared with that expected value.
The difference between the measured and expected values is called mass error. Depending on the instrument and analytical method, mass error may be reported in units such as daltons or parts per million (ppm).
A close match between the observed and expected mass can support the identification of a peptide. However, the result should still be considered alongside the analytical method and other available data.
How Reference Data Helps
Researchers can compare an experimental spectrum with previously measured reference spectra. This can provide additional information beyond simply comparing an observed mass with a theoretical value.
Peptide spectral libraries contain annotated reference spectra that researchers can search when evaluating acquired MS/MS data.
Reference spectra can be useful because they contain information about the actual pattern of fragment ions measured from a peptide, rather than only its expected molecular mass.
What Mass Spectrometry Can and Cannot Establish
Mass spectrometry can provide information about molecular mass and the ions detected in a sample. When MS/MS is performed, the resulting fragment pattern can provide additional information that can support a proposed peptide identification and sequence analysis.
At the same time, an MS result should not automatically be treated as a complete description of a research material. Mass spectrometry provides specific types of information, while other analytical methods may provide information that MS does not directly measure.
For example, chromatography can provide information about separated components, while other analytical methods may be used to examine additional physical or chemical properties. Different analytical methods can answer different questions about a sample; the conclusions depend on the tests performed and their limitations.
How Mass Spectrometry Fits Into Peptide Analysis
Mass spectrometry is one part of a broader analytical process. Depending on the testing performed, a peptide report may include an observed mass, expected mass, chromatographic results, MS/MS data, or other measurements.
When reviewing an MS result, researchers can ask:
- What type of mass spectrometry was performed?
- What does the reported m/z value represent?
- Was the observed mass compared with an expected value?
- Was MS/MS performed?
- Was the spectrum compared with reference data?
- What other analytical results are included?
Looking at these details together can provide more context than relying on a single mass value or peak.
Use the product name and batch or lot information to locate the applicable documentation in the Grey Axis COA library. Review the report itself to determine whether MS, MS/MS, or LC-MS was performed and what the testing laboratory reported.
View the Grey Axis COA Library
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