Molecular weight analysis by MS-MALDI TOF

Molecular mass determination by MS-MALDI TOF of biological or synthetic samples, such as:
Proteins
Peptides
Glycoproteins and glycopeptides
Oligosaccharides
Oligonucleotides
Natural or synthetic polymers
Synthesis products

The objective is to obtain the mass spectrum with the exact mass of peptide and/or protein. This analysis is used for natural or synthetic peptide and for recombinant or purified proteins.
Matrix Assisted Laser Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) allows analysis of proteins and peptides with high sensitivity and soft ionization with little or no fragmentation. Ions are formed through gas-phase proton-transfer reactions with photoionized matrix molecules. Matrix is typically a small energy absorbing molecule such as 2, 5-dihydroxybenzoic acid (for synthetic peptides) or sinapinic acid (for intact proteins). MALDI-TOF MS analysis produces data for peptide and protein molecular weight determination.
MALDI-MS is also sensitive to the presence of contaminants such as salts or surfactants, and protein-containing solutions that have high levels of these contaminants often exhibit little or no protein signal. Such contamination intrinsically results from some purification procedures, and in certain cases, salts or surfactants are required to keep proteins from precipitating from solution. Contaminants likely reduce MALDI-MS signals by disrupting the cocrystallization of analytes and matrix molecules.
To avoid this problem we recommend:
1. Dyalisis of the sample.
2. Submit the sample as concentrated as possible to allow us to be diluted in the appropriate solvent.
3. Desalt the sample by zip-tip, oasis, omix¿(pipette tips/columns containing C18 or C4 media)
The amount needed for the analysis of peptides is the range of femtomoles and for proteins is the range of picomoles.
Procedure:
An aliquot (0.5 ul) of the sample (protein/peptide) is analyzed by a MALDI-TOF-type of mass spectrometry using an Autoflex model of Bruker Daltonic (Bremen, Germany), in a positive ion reflector mode, and employing (0.5 ul) sinapinic acid for proteins and 2,5-dihydroxybenzoic acid (0.5 ul) for peptides as matrix and an Anchor-Chip surface target (Bruker Daltonic). Peak identification and monoisotopic peptide mass assignation were performed automatically using Flexanalysis¿software, version 2.2 (Bruker Daltonics).

Precise determination of the molecular weight of peptides and proteins. For peptides of less than 5 kDa, mass errors are below 50 ppm in reflector mode. Molecular masses of proteins of up to 70 KDa can be measured in linear mode. Although 70 kDa is not the mass limit of the instrument analysis of proteins of proteins higher than 50 KDa becomes increasingly difficult. Mass error also increases with mass, being of up to 200 ppm at 10 KDa.
Fairly complex peptide or protein mixtures can be analyzed without previous separation (The technique can be used to obtain proteome fingerprints which is the basis of protein profiling technologies such as SELDI).
Samples must be delivered dissolved in a buffer, preferably a volatile one, with a salt concentration lower than 50 mM.

Molecular weights (MW) and selected mass fragmentation (MSMS) are determined by MALDI-TOF mass spectrometry.
This type of analysis is carried out from samples in solution at known concentrations (or lyophilized samples in estimated quantities). Samples can be proteins, peptides, DNA and RNA oligonucleotides, PNAs and other types of biological or synthetic polymers. Depending on the type of sample, different matrices are used that co-crystallize with it.
The analysis includes sample dilution to the optimum concentration for analysis, acquisition of mass spectra in the specified mass range and a report of results in which the procedure followed is indicated and the obtained spectra, mass list and intensities are included.
It is recommended that the samples be dialyzed or dissolved in a volatile buffer and as concentrated as possible, so that they can be diluted with suitable solvents. In the case of samples containing components (surfactants, salts, etc.) that interfere with the crystallization process, the corresponding variant will be selected and desalination and concentration will be carried out to eliminate the interferences.
For the analysis of proteins and oligonucleotides, quantities of the picomoles/ul range and in the case of peptides, femtomoles/ul are required. A minimum volume of about 5 ul will be required. The larger the size of the molecules to be analyzed, the greater the requirement for sample quantity.
For molecules between 500 Da and 5 KDa, analysis will be carried out in reflector mode, with an estimated error of 10 ppm. For molecules larger than 5KDa, linear mode will be used, whose estimated error is 0.01% (100 ppm).
INSTRUCTIONS FOR SUBMITTING SAMPLES
1.- The sample in solution or lyophilized must be sent in a 0.5 ml tube clearly identified with the sample name labeled on the lid.
2.- Samples will be delivered or sent along with the corresponding application properly filled out (MOLECULAR WEIGHT DETERMINATION AND FRAGMENTATION REQUEST), where the concentration and solvent composition (protein quantity of dried or lyophilized samples) must be specified.
3.- It is important to take into account the different contaminants that can interfere in the process of analysis by mass spectrometry (INTERFERENTS).
NOTES:
If it is a protein sample in gel, it must be previously extracted from it.

In the determination of molecular masses of compounds in solution by matrix-assisted laser desorption ionisation time-of-flight (MALDI-TOF) mass spectrometry , the compounds are co-cristalizated on a stainless steel target with a given matrix. Usually it is used acid Sinapinico (SA), acid dihydroxybenzoic acid (DHB) or alpha-cyano-hydroxy-cinnamic acid (HCCA) depending on the compound whose mass is to be determined. HCCA and DHB are used in the case of peptides and SA with proteins. The energy required for the ionization of the compounds is provided by a laser. The matrix crystals absorb laser energy and convert it to heat leading to localized "explosion" of the sample/matrix (the plume jet). Proton transfer in the resulting plasma yields ions (MH +) and being in the gas phase these ions are accelerated by an electric field into the analyzer (time of flight) and are sorted according to their relationship to mass/charge (m/z).

Molecular mass determination of biomolecules (proteins, peptides, oligonucleotides, sugars ...) and large organic molecules (such as polymers, dendrimers, polyphenols and other macromolecules) by MALDI-TOF/TOF mass spectrometry.
Samples must be in solution. Depending on the molecular mass (small 20,000 Da), the concentration necessary for the analysis could vary from few femtomoles to hundreds of picomoles. The solution should be free of salts, detergents and other contaminants which may impede or hinder the crystallization of the sample with the matrix.
In the case of special samples, and if the user agrees, studies with different solvents and matrix types will be made in order to optimize the results.