Molecular mass fingerprints

Key Words Invertebrate immunity antimicrobial peptides immune effectors mass spectrometry drug discovery Drosophila arthropods peptide purification molecular mass fingerprints bioactive peptides. 

Purity control, molecular mass determination and molecular mass fingerprints can be performed either by electrospray ionization (ESI) or matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) MS. ESI is preferred for sequence determination by MSn and MALDI-TOF for molecular mass fingerprints. Both technologies are appropriate for molecular mass determination and purity control. We have edited a critical review on the use of MS strategies for discovery and peptide sequencing of bioactive peptide (25). 

Stop the reaction by addition of 1 volume of an acidic solution (e.g., 0.1% TFA) and control the pH using pH paper (pH < 4). For direct molecular mass fingerprint see Note 13. 

For molecular mass fingerprint of an enzymatic digestion, the acidity of the matrix and of the 1% TFA droplet is sufficient to quench any further digestion. 

Then the mass spectrum of the mentioned peptide mixture is measured. The set of molecular mass values (peak list) corresponding to individual peptides is characteristic for the protein and can be considered as its fingerprint. 

Different authors used RP-HPLC and UV detection to monitor peptide formation during cheese ripening [174-178], providing valuable information about proteolysis. When large hydrophobic peptide need to be separated an lEC represents the best choice [179]. Nevertheless, the identification of these peptides is essential for the complete understanding of the proteolytic process. The peptides eluted from the LC column can be subjected to ESl-MS for molecular weight determination and MS/MS for amino acid sequence determination, which allow rapid peptide identification [172]. HPLC-ESl-MS and MS/MS techniques have been successfully used for peptide mass fingerprint purposes for sequence analysis of purified albumin from Theobroma cacao seeds [180,181]. 

Characterization of noncovalent bonding of the proteins can also be done using MS. For example MALDI MS has been used in measurement of the molecular mass of the noncovalendy linked tetramer of glucose isomerase, a complex consisting of identical monomers of 43.1 kDa each. MALDI-TOFF peptide mass fingerprinting combined with electrospray tandem mass spectrometry can efficiently solve many complicated peptide protein analysis problems. 

The OPCW requires two independent techniques for identification. In the case of GC/MS, El, and Cl spectra, acquired from separate chromatographic runs, are regarded as independent techniques. A combination of the two is accepted as unequivocal identification - one as a fingerprint, the other to confirm molecular mass. LC/MS provides an alternative to GC/CI/MS for confirmation of the molecular mass and LC/MS/MS provides a partial fingerprint. However, LC/MS/MS spectra, which are normally acquired under identical chromatographic conditions as LC/MS spectra, are not currently considered as a second independent identification. Further development of these criteria may be required with instrumental development. LC retention time is accepted as a second technique if the retention time falls within a window of 0.2 min of the retention time for an authentic chemical, with a signal-to-noise ratio of at least 5 1. However, great care has to be exercised if identification is based solely on LC/MS. 

The profile of the masses of the peptides obtained by one of these methods is compared by means of a computer with all predicted peptide digests from a database of proteins to identify the best possible matches. This is termed peptide mass fingerprinting (PMF). A protein generally can be identified using the mass of four to six of its cleavage peptides having masses in the range 700-3000 Da and determined with an accuracy of 0.05 to 0.0005 %. This is improved further if the molecular mass of the protein also is provided [83-86],... 

Peptide mass-fingerprinting of the I-labeled PBPs (Fig. la) revealed the eight known PBPs of E. coli (Thble 2). Unlike SDS-PAGE analysis of labeled PBPs (19), the elution order by RPLC is not related directly to molecular weight but is dictated by hydrophobicity. 

The approaches described in the previous section enable the molecular-mass determination of intact proteins, generally with an accuracy better than 0.01%. Further stractural characterization of the protein requires determination of possible post-translational modifications (PTM) as well as the amino acid sequence. In addition, issues related to tertiary and quaternary stracture of the protein, the presence of cofactors, etc., may be relevant. LC-MS plays an important role in the primaiy and secondaiy stractural characterization of proteins, i.e., in terms of amino-acid sequencing and PTM. The procedure generally involves chemical or enzymatic treatment of the intact protein, acquisition of a peptide map or peptide mass fingerprint by either direct infusion (nano-)ESI-MS or RPLC-MS, and the amino-acid sequencing of individual peptides by means of product-ion MS-MS. Further experiments may be needed in relation to PTM, as outlined in more detail in Ch. 19. 

The efficacy of MS to characterize effectors of Drosophila immunity and Phormia (also referred in the literature as Protophormia) terranovae is illustrated by several examples. This will not follow a chronological order but is in accordance with the complexity of the MS analyses. MS allowed (i) to define precise molecular masses, to identify post-translational modifications (N-terminal cyclisation, C-terminal amidation, disulfide bonds array, glycosylation), (ii) to determine primary structures of immune peptides (sequencing by MS/MS), (iii) to have mass fingerprints and to identify peptide effectors that are part of Drosophila immunity (molecular mass differential display by MALDI-MS),... 

Nevertheless, the authors observed that several molecular masses between 3-5 kDa were detected exclusively in the fat body tissue collected from bacteria-challenged larvae of this flesh-fly while others were only present in control flies [55]. Such approach evidenced the sensitivity and potency of MALDI-MS to perform peptide mass fingerprints directly on tissues or organs. Analyzing complex biological samples by MALDI-MS without any pretreatment of the sample e.g. solid-phase extraction, LC) has thus become a reality. 

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