Mass Spectrometry

MALDI

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.

1A 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 a 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
• Certain peptides can be selected for and given higher energies causing them to fragment providing some sequence information.

• 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 the number of matching fragments and the probability that the matching is not a random event.
• PMF is the method-of-choice for protein identification in proteome studies because it is a simple and sensitive technique with an ever increasing level of confidence due to improved mass accuracy and availability of more complete genomic information for a growing number of organisms.

• 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 MS-MS analysis specific peptide ions are selected 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. Ions can also pass through a collision chamber containing gas molecules whereby the collisions of the ions with these ions can induce further fragmentation.
• 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.

1b Some points to note:

• the x axis of the spectra represents m/z - that is mass divided by charge. In MALDI-ToF, ions are nearly always singly charged [M+H]+ species (where M represents the molecule of interest; and H is hydrogen). To obtain an exact Molecular Weight, it is therefore a matter of subtracting the mass of a single hydrogen (1.0079) from any mass shown on a spectrum.
• The data generated will fall in the m/z range of 800-4000 Da for Peptide Mass Fingerprinting. All the peaks that observed on the mass spectrum are put into a mass list and it is these that are used to search against a database. Database searches are performed using web based tools such as MASCOT or MS-Fit.
• An expanded view of the peaks reveals a group of peaks differing by 1 Dalton which is called an isotope distribution. This is caused by naturally occurring Carbon-13 (13C has an abundance of approximately 1% of 12C). As any given peptide contains many carbon atoms then the contribution from 13C can be significant. The peak with the lowest molecular weight is the 'monoisotopic' peak, and only contains 12C. The remaining peaks contain one more or more 13C atoms each increasing the apparent mass by 1Da. All mass lists s upplied will be listing the monoisotopic peaks only.
• In the interests of clearer database searching, a certain number of masses are deliberately excluded from the mass list supplied. Peaks resulting from autolysis of trypsin, from commonly occurring keratin, and from any salt adduct peaks are removed.
• 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.

1c Typical Procedure

1. Protein spots from coomassie / silver stained gels.
2. Tryptic digest with desalting.
3. MALDI-MS to obtain mass list.
4. Database search MSMS
5. MS-MS analysis identification confirmation.
6. 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.

Example MSMS spectra

1d Sample Information 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.
• Investigators may also elect to perform their own sample analysis in which case raw data can be forwarded without further analysis though some guidelines and advice can be given for database searching.

1e Sample preparation

• Salt or buffers at concentrations >10mM
• 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

• 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.