

Page contents

  • Introduction to Mass Spectrometry
  • Mass Spectrometry Applications
  • Quantitative Proteomics
  • Sample Preparation

Introduction to Mass Spectrometry

Proteomics is the study of all proteins in a biological system (e.g., cells, tissue, organism) during specific biological events (1, 2).  The complementation of genomics and proteomics is considerably more difficult to study than genomics or even transcriptomics alone because of the dynamic nature of protein expression. Additionally, the majority of proteins undergo some form of post-translational modification (PTM), further increasing proteomic complexity. The broad scope of proteomics has only begun to be realized within the last 15 years due in large part to technological developments in mass spectrometry.

Mass spectrometry is a sensitive technique used to detect, identify and quantitate molecules based on their mass and charge (m/z) ratio. Originally developed almost 100 years ago to measure elemental atomic weights and the natural abundance of specific isotopes (3), MS was first used in the biological sciences to trace heavy isotopes through biological systems; later on, MS was used to sequence oligonucleotides and peptides and analyze nucleotide structure (4).

The development of macromolecule ionization methods, including electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI), enabled the study of protein structure by MS (5, 6, 7). This allowed scientists to obtain protein mass "fingerprints" that could be matched to proteins and peptides in databases to predict the identity of unknown proteins. Methods of isotopic tagging have led to the quantitation of target proteins both in relative and absolute quantities. Technological advancements have resulted in methods that analyze samples in solid, liquid or gas states. The sensitivity of current mass spectrometers allows one to detect analytes at concentrations in the attomolar range (10-18) (10).

Mass Spec Analysis Technical Handbooks

All the tools you need to optimize your mass spec experiments

  • Handbook 1: Thermo Scientific 2015 New Product Guide for Mass Spectrometry
  • Handbook 2: Thermo Scientific Pierce Controls and Standards for Mass Spectrometry
  • Handbook 3: Thermo Scientific Pierce Products for Mass Spectrometry Sample Preparation
  • Handbook 4: Thermo Scientific Pierce Reagents for Quantitative Proteomics

Mass Spectrometry Applications


This overview outlines the role of mass spectrometry in the field of proteomics and reviews MS methodology and instrumentation. It also touches on sample preparation and liquid chromatography-based separation prior to MS analysis.

Common applications and fields that use mass spectrometry
Field of Study Applications
  • Determine protein structure, function, folding and interactions
  • Identify a protein from the mass of its peptide fragments
  • Detect specific post-translational modifications throughout complex biological mixtures
  • Quantitate (relative or absolute) proteins in a given sample
  • Monitor enzyme reactions, chemical modifications and protein digestion
Drug Discovery
  • Determine structures of drugs and metabolites
  • Screen for metabolites in biological systems
Clinical Testing
  • Perform forensic analyses such as confirmation of drug abuse
  • Sequence oligonucleotides
  • Test water quality or food contamination
  • Measure petroleum composition
  • Perform carbon dating

All mass spectrometers have an ion source, a mass analyzer and an ion detector. The nature of these components varies based on the type of mass spectrometer, the type of data required, and the physical properties of the sample. Samples are loaded into the mass spectrometer in liquid, gas or dried form and then vaporized and ionized by the ion source (e.g., APCI, DART, EI).

Schematic of the basic components of a mass spectrometer.
Schematic of the basic components of a mass spectrometer.


Ions that have successfully been deflected by the mass analyzers then hit the ion detector. Most often, these detectors are electron multipliers or microchannel plates, which emit a cascade of electrons when each ion hits the detector plate (4). This cascade results in amplification of each ion hit for improved sensitivity. This entire process is performed under an extreme vacuum (10-6 to 10-8 torr) (8) to remove gas molecules, neutrals, and contaminating non-sample ions, which can collide with sample ions and alter their paths or produce non-specific reaction products (9).

Newer orbitrap technology captures ions around a central spindle electrode and then analyzes their m/z values as they move across the spindle with different harmonic oscillation frequencies. Orbitrap technology can achieve extremely high sensitivity and resolution of obtained mass spectra.

Mass spectrometers are connected to computers with software that analyzes the ion detector data and produces graphs that organize the detected ions by their individual m/z and relative abundance. These ions can then be processed through databases to predict the identity of the molecule based on the m/z.

Diagram of a sector mass spectrometer.

Tandem mass spectrometry (MS/MS) offers additional information about specific ions. In this approach, distinct ions of interest are selected based on their m/z from the first round of MS and are fragmented by a number of methods of dissociation. One such method involves colliding the ions with a stream of inert gas, which is known as collision-induced dissociation (CID) or higher energy collision dissociation (HCD). Other methods of ion fragmentation include electron-transfer dissociation (ETD) and electron-capture dissociation (ECD).

These fragments are then separated based on their individual m/z ratios in a second round of MS. MS/MS (i.e., tandem mass spectrometry) is commonly used to sequence proteins and oligonucleotides because the fragments can be used to match predicted peptide or nucleic acid sequences, respectively, that are found in databases such as IPI, RefSeq and UniProtKB/Swiss-Prot. These sequence fragments can then be organized in silico into full-length sequence predictions.

Diagram of tandem mass spectrometry (MS/MS).


Gas chromatography (GC) and liquid chromatography (LC) are common methods of pre-MS separation that are used when analyzing complex gas or liquid samples by MS, respectively. LC-MS is typically applied to the analysis of thermally unstable and nonvolatile molecules (e.g., sensitive biological fluids), while GC-MS is used for the analysis of volatile compounds such as petrochemicals. LC-MS and GC-MS also use different methods for ionization of the compound as it is introduced into the mass spectrometer. With LC-MS, electrospray ionization (EI) is commonly applied, resulting in the production of aerosolized ions. With GC-MS, the sample may be ionized directly or indirectly via EI.

High performance liquid chromatography (HPLC) is the most common separation method to study biological samples by MS or MS/MS (termed LC-MS or LC-MS/MS, respectively), because the majority of biological samples are liquid and nonvolatile. LC columns have small diameters (e.g., 75 μm; nanoHPLC) and low flow rates (e.g., 200 nL/min), which are ideal for minute samples (2). Additionally, "in-line" liquid chromatography (LC linked directly to MS) provides a high-throughput approach to sample analysis, enabling multiple analytes to elute through the column at different rates to be immediately analyzed by MS. For example, 1-5 peptides in a complex biological mixture can be sequenced per second by in-line LC-MS/MS (2).

Example of in-line LC-MS/MS system.

Quantitative Proteomics


Relative quantitation strategies include stable isotope labeling using amino acids in cell culture (SILAC) and tandem mass tagging (TMT). In these approaches, proteins or peptides are labeled with stable isotopes that give them distinct mass shifts over unlabeled analytes. This mass difference can be detected by MS and provides a ratio of unlabeled to labeled analyte levels. These approaches are often used in discovery proteomics, where many proteins are identified across a broad dynamic range using different-sized labels.


Sample Preparation



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