Amino acids isobaric

The primary goal of peptide mapping is the verification of the amino acid sequence deduced from the genetic code of the recombinant protein. The protein backbone gets cleaved by typically two or three different endoproteinases like Lys-C, trypsin, and Glu-C to achieve maps with sequence-overlapping peptide fragments. These peptide mixtures can then be separated by LC or CE and analyzed on-line by MS to obtain sequence information. Often simple mass analysis matches the predicted primary sequence of the protein. However, sometimes mutations can lead to isobaric masses of peptides that can be overseen, if no further sequence analysis like N-terminal Edman sequencing and MS/MS is carried out. 

Frequently a third enzymatic digestion is necessary to eliminate all the possible ambiguities. Using trypsin as a proteolysis enzyme allows one partially to distinguish Lys from Gin, two isobars. In fact, the trypsin specifically cleaves on the C-terminal side of Lys and Arg, so all the amino acids with mass 128 ending a peptide can be identified as Lys. However, the absence of a cleavage cannot be used as proof in identifying an amino acid... 

ESI spectrum of a peptide library TQTXT. The letters correspond to the amino acid in position X. Isobaric amino acids (Q/K) and 13C-isobaric amino acids (L/N, N/D and Q/E) cannot be distinguished at the low resolution used. The difference in abundance between the peaks is due to the different response factors and suppression effects. For this reason, quantification of peptide libraries based on a simple mass measurement is not always possible. Reproduced (modified) from Schlosser G., Takats Z., Vekey G., Pocsfalvi G., Malorni A., Windberg E., Kiss A. and Hudecz F., Journal of Peptide Sciences, 9, 361-374, 2003, with permission. 

De novo sequencing seldom leads to a complete sequence becanse in most cases the y- and b-ion series are not completely present, some sequence ions might be lost in the noise, and/or ambiguities exist due to additional peaks (neutral losses of CO, HjO, NH3, etc.), and isobaric and isomeric amino acids (Table 17.2). 

Metabolites are small molecules that participate as substrates or products in metabolic reactions essential for the normal function of a cell. This molecular class comprises a wide range of compounds, from amino acids to lipids, organic acids, and nucleotides (11). The wide range of concentration and different chemical properties make the analysis of these compounds a challenging task. Usually, high-resolution mass spectrometers and chemical derivatization strategies are necessary to resolve isobaric interferences, increase ionization efficiency, and overcome chemical background effects from the matrix-assisted laser desorption/ionization (MALDI) matrix or the tissue matrix itself (12). 

Not only are forensic investigations of suspected AN poisonings frequently hampered in its detection due to its rapid decay but the widespread presence of the amino acid, Phe, also presents major analytical problems (Gugger et al., 2005). Since these compounds are isobaric (both molecular weights = 165) and elute similarly in liquid chromatography, misidentification of AN is possible. Approaches to prevent the misidentification of AN that have been explored in these studies included... 

A number of low-molecular-weight A-acyl amino acid lipids of biological significance were identified based on values of t and miz values provided by IMS-MS. Both data acquisition and visnalization of the separated isomeric lipids is rapidly achieved. For example, A-arachidonoyl isoleucine, and A-arachidonoyl leucine exhibit t fingerprints that are sufficiently distinct to delineate isomeric composition. It is often difficult to obtain meaningful MS/MS fragment ions from these low abundant lipids for structural characterization. The mass window for parent ion selection for most MS instruments is insufficient to cleanly select isobaric compounds, and the fragmentation pattern of isomers is commonly very similar. 

Jensen and co-workers showed that isobaric tags can be used in combination with 2D-PAGE and affinity purification using Fe(III)-IMAC (immobilized metal ion affinity chromatography) to quantify differences in phosphorylation states. They were able to quantify the relative amounts of a peptide with one phosphate at different amino acid positions by HPLC, because the three isoforms have different retention times. 

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