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Mass Spectrometry

MALDI TOFTOF

Applied Biosystems 4700 Proteomics Discovery System

This Matrix-Assisted Laser Desorption Ionization Time-of-Flight Time-of-Flight (MALDI-TOF-TOF) system provides high quality data for comprehensive proteomic applications with high throughput capability.

Theory

  • The peptides are co-crystrallised with matrix molecules which facilitates ionization after high energy pulses of the laser.
  • The ions are accelerated by a high electric potential, enter the TOF chamber under high vacuum, and differences in mass are separated according to the time taken to reach the detector.
  • The time-of-flight is directly proportional to the mass-to-charge ratio of an ion, and a mass spectrum is obtained.
  • In MALDI-TOF-MS peptides generally carry one charge and consequently each isomeric form gives only one peak in a spectrum
  • In TOFTOF analysis, specific peptide ions are selected for and passed through the second TOF chamber. These ions fragment after having acquired sufficient internal energy which is released by undergoing fragmentation into defined product ions, a process known as post source decay (PSD). Further information can be obtained by passing ions through a collision chamber containing gas molecules such that ion-gas collisions induce further fragmentation, a process known as collision induced decay (CID). By this method Immonium ions are generated revealing information of the amino acid composition of the peptide. 
  • The combination of a MALDI TOF analysis with a HPLC separation with online fraction spotting produces a technique with excellent sensitivity as all salts and interfering substances are removed minimising signal suppression.

 Typical Experiments and Procedures

Protein Identification: Peptide Mass Fingerprinting

  • After tryptic digestion the mass spectra obtained of the resulting peptide fragments produces a mass list that can be used to interrogate a database of known proteins that have been theoretically digested with trypsin.
  • This procedure is known as peptide mass fingerprinting (PMF).
  • Positive hits are based on matching a number of fragments and supported by sequence information obtained by TOFTOF analysis.
  • This method obtains a peptide fragmentation spectrum which is specific for an individual selected peptide and the complete or partial sequence can be elucidated.
  • In TOFTOF analysis, a selection of peptides ions are selected for and fragmented yielding sequence information which can be added to the database search. This increases the confidence of the match if the TOFTOF fragments matches those expected from the same sequence of the identified protein.
  • Peptides can be selected from the same sample used to generate a peptide mass fingerprint; so there is no additional peptide or protein material required.
  • Example of protein identification from acquisition of the tryptic digest to selection and fragmentation of various peptides matched to the database search and protein identification result.
Example MSMS spectra
  • PMF combined with TOFTOF sequencing is the first method-of-choice for protein identification in proteome studies because it is a fast, simple and sensitive technique.
  • We recommend this technique when:
    - The sample contains low concentrations of salts or buffers.
    - Your samples are relatively simple and clean - 1 or 2 proteins (ie. 2D gel spot).
    - You are working with organisms with a well-characterized genome.
    - You would like to get your answer fast
    - For low level samples or complex samples LCMALDI would be the method of choice.

Peptide Mass Determination

  • In reflectron mode a high level of resolution and accuracy is achieved to yield a fast and effective technique for a simple mass determination.
  • The tolerance of MALDI to the presence of salts reduces the potential amount of sample preparation required for analysis, although salts and other ionisable molecules can affect the sensitivity of detection.

Protein Mass Determination

  • In linear mode we obtain an almost limitless range of masses that we can analyse.
  • Limitations of resolution in linear mode significantly reduces the mass accuracy. However it does represent a fast and simple method for the detection and QC of a protein.
  • MALDI analysis of a protein can be particularly useful when there is significant heterogeneity in PTM's such as glycosylation, as it produces a clear average mass of all species.
  • As with peptide analysis, the presence of salts and buffers affects the sensitivity of detection however in low to moderate concentrations a mass is still often observed reducing the need for sample desalting which often mean significant protein losses.

Protein Quantitation: Isobaric mass tagging

  • The use of isobaric tags such as ITRAQ, ICAT, Silac, Exactag etc allows relative the quantitation of individual proteins from two different samples treated with a different tag.
  • Quantitation determined by measuring the relative intensity of reporter fragments in the TOFTOF spectra.

Sample Information and Preparation Required

  • Sample origin and species are helpful in making protein assignments based on database searching results.
  • Knowledge of sample buffer is important because not all buffers are compatible with mass spectrometry.
  • This is especially important if you are providing your sample in solution rather than in a gel.
  • If you are providing a gel, please indicate what the gel is stored in, known and possible modifications (i.e. cys alkylation, phosphorylation) are also very important in obtaining accurate results from database searching.
  • Keratin can be a significant problem when dealing with protein identification. Contamination can occur at many different points in processing your sample: pouring the gel, staining the gel, cutting out protein spot or band from the gel, or just in general sample handling. The best way to avoid potential keratin contamination is to wear gloves and a lab coat when working with your sample. Any efforts to decrease the possibility of keratin contamination will improve your results from the mass spectrometer.
  • For optimal results non-gel based samples must not contain :
    - Salt or buffers at concentrations >50mM
    - Organics such as DMF, DMSO
    - Samples that contain Na, K, Ca, Li, or PO4 as these can form adducts complicating a spectra.
    - Samples containing detergent or stabilizers such as PEG or glycerol
    - High concentrations of organic solvents
    - Samples that contain significant particulate or insoluble material
    - Radioactive or biohazardous samples
  • We recommend:
    - Eppendorf brand tubes rinsed with MeOH and dried prior to use.
    - The use of volatile buffers such as H2O, MeOH, Acetonitrile, Ammonium Bicarbonate, Acetic acid, Formic acid, Trifluoroacetic acid.
    - All reagents should be of the highest quality available.
    - No colored eppendorf tubes! These tubes often contain residual quantities of dyes which have been found to produce peaks in spectra. Since organic solvents are used in sample preparation of mass spec, the dyes can leach off the tubes into your samples. Use only clear plastic tubes.