The objective of this study was to determine if liquid chromatography mass spectrometry (LC/MS) data of tryptic digests of proteins can be used for quantitation. In theory, the peak area of peptides should correlate to their concentration; hence, the peak areas of peptides from one protein should correlate to the concentration of that particular protein. To evaluate this hypothesis, different amounts of tryptic digests of myoglobin were analyzed by LC/MS in a wide range between 10 fmol and 100 pmol. The results show that the peak areas from liquid chromatography mass spectrometry correlate linearly to the concentration of the protein (r2 = 0.991). The method was further evaluated by adding two different concentrations of horse myoglobin to human serum. The results confirm that the quantitation method can also be used for quantitative profiling of proteins in complex mixtures such as human sera. Expected and calculated protein ratios differ by no more than 16%. We describe a new method combining protein identification with accurate profiling of individual proteins. This approach should provide a widely applicable means to compare global protein expression in biological samples.

Deamidation of asparagine residues of biological pharmaceuticals is a major cause of chemical degradation if the compounds are not formulated and stored appropriately. The mechanism of this nonenzymatic chemical reaction has been studied in great detail; however, the identification of deamidation sites in a given protein remains a challenge. In this study, we identified and characterized all deamidation sites in the conserved region of a recombinant monoclonal antibody. The conserved region of this antibody is shared by all human IgGs with the exception of minor differences in the hinge region. Our high-performance liquid chromatography method could separate the succinimide, isoaspartic, and aspartic acid isoforms of peptide fragments generated using trypsin. Each of the isoforms was unambiguously identified using tandem mass spectrometry. Deamidation at the identified four sites was slow for the intact, folded antibody at accelerated degradation conditions (pH 7.5 and 37 degrees C). Deamidation was enhanced after reduction, alkylation, and tryptic digestion, indicating that the three-dimensional structure of the antibody reduced deamidation. Furthermore, after the reduction, alkylation, and tryptic digestion, only 4 of a possible 25 asparagine residues showed deamidation, demonstrating the effect of the primary amino acid sequence, especially the -1 and +1 amino acids flanking the deamidation site. For instance, the amino acid motifs SNG, ENN, LNG, and LNN were found to be more prone to deamidation, whereas the motifs GNT, TNY, YNP, WNS, SNF, CNV, SNT, WNS, FNW, HNA, FNS, SNK, GNV, HNH, SNY, LNW, SNL, NNF, DNA, GNS, and FNR showed no deamidation. Our findings should help predict deamidation sites in proteins and peptides and help develop deamidation-resistant biological therapeutics.

In this report, we describe an approach for identification and relative quantitation of individual proteins within mixtures using LC/MS/MS analysis of protein digests. First, the proteins are automatically identified by correlating the tandem mass spectra with peptide sequences from a database. Then, peak areas of identified peptides from one protein are added together to define the total reconstructed peak area of the protein digest. The total reconstructed peak area is further normalized to the peak area of an internal standard protein digest present in the mixture at a constant level. The method was illustrated using digested mixtures of five standard proteins as follows. One protein was gradually diluted while the other four components were present in the mixtures at constant level. This study revealed that relative peak area of the variable protein increased linearly (trend line R2 = 0.9978) with increasing amount from 10 to 1000 fmol, while relative peak areas of four constant proteins remained approximately the same (within 20% relative standard deviation). To further evaluate the applicability of this method for the quantitation of proteins from complex mixtures, human plasma protein digest was spiked with 200 and 400 fmol of myoglobin digest. Total peak area of myoglobin peptides was normalized to the total peak area of apolipoprotein A-I peptides from human plasma, which played the role of an internal standard. The myoglobin/apolipoprotein A-I peak area ratio was 2 times larger for the human plasma digest spiked with a double amount of myoglobin. After several repetitions, the error of the relative peak area measurements remained below 11%, suggesting that the method described here can be used for relative concentration measurements of proteins in the complex biological mixtures. In the presented method, chemical derivatization steps are not needed to create an internal standard, as in isotope-coded affinity tag or similar methods.

The status of the N-terminus of proteins is important for amino acid sequencing by Edman degradation, protein identification by shotgun and top-down techniques, and to uncover biological functions, which may be associated with modifications. In this study, we investigated the pyroglutamic acid formation from N-terminal glutamic acid residues in recombinant monoclonal antibodies. Almost half the antibodies reported in the literature contain a glutamic acid residue at the N-terminus of the light or the heavy chain. Our reversed-phase high-performance liquid chromatography-mass spectrometry method could separate the pyroglutamic acid-containing light chains from the native light chains of reduced and alkylated recombinant monoclonal antibodies. Tryptic peptide mapping and tandem mass spectrometry of the reduced and alkylated proteins was used for the identification of the pyroglutamic acid. We identified the formation of pyroglutamic acid from N-terminal glutamic acid in the heavy chains and light chains of several antibodies, indicating that this nonenzymatic reaction does occur very commonly and can be detected after a few weeks of incubation at 37 and 45 degrees C. The rate of this reaction was measured in several aqueous buffers with different pH values, showing minimal formation of pyroglutamic acid at pH 6.2 and increased formation of pyroglutamic acid at pH 4 and pH 8. The half-life of the N-terminal glutamic acid was approximately 9 months in a pH 4.1 buffer at 45 degrees C. To our knowledge, we showed for the first time that glutamic acid residues located at the N-terminus of proteins undergo pyroglutamic acid formation in vitro.

Succinimide formation increased as pH became more acidic, whereas its hydrolysis was faster as pH became neutral and alkaline. Succinimide hydrolysis in a denatured sample was estimated to have completed in less than 2 h, but approximately three days for a similar pH but without denaturant. These observations suggest that protein conformation affects succinimide hydrolysis.