Increasing clinical liquid chromatography/tandem mass spectrometry assay throughput using a full calibration curve generated by one injection from a single-tube calibrator.

Mass spectrometry (MS) generally delivers more accurate results than immunoassay (IA) for certain clinically relevant analytes, but IA is still the more prevalent methodology used by clinical laboratories because of barriers to MS adoption, such as lower throughput. Therefore, it is increasingly important to develop new strategies to increase LC/MS/MS throughput so that more accurate results can be delivered to patients and clinicians. METHODS: Throughput can be increased by reducing assay calibration time using a single-tube calibrator, a mix of isotopologues of the target analyte at different concentrations in a biological matrix, rather than a set of traditional, multiple-tube calibrators. One injection from a single-tube calibrator can generate a full calibration curve such that each calibration point is from the multiple reaction monitoring (MRM) signal corresponding to a specific isotopologue. RESULTS: In this study, a single-tube calibrator (five levels in one vial) and a set of multiple-tube calibrators (seven levels in seven vials) were used to measure the concentration of testosterone in 42 serum samples originally value assigned by the Centers for Disease Control and Prevention (CDC) reference method. The bias between the CDC reference method and the single-tube calibrator measurements and the multiple-tube calibrators measurements was +1.1% and - 5.5%, respectively. These results were within the CDC Hormone Standardization (HoSt) program bias acceptance criteria of ±6.4%. CONCLUSIONS: The results show that LC/MS/MS throughput can be increased using a single-tube calibrator because it reduces assay calibration time while delivering equivalent results to those generated using traditional, multiple-tube calibrators. The single-tube calibrator may also save cost to laboratories through reductions in consumable consumption, technician labor time, and inventory management, as well as to manufacturers because fewer vials would need to be manufactured, tested, stored, and shipped.

Dataset and standard operating procedure for newborn screening of six lysosomal storage diseases: By tandem mass spectrometry.

In this data article we provide a detailed standard operating procedure for performing a tandem mass spectrometry, multiplex assay of 6 lysosomal enzymes for newborn screening of the lysosomal storage diseases Mucopolysaccharidosis-I, Pompe, Fabry, Niemann-Pick-A/B, Gaucher, and Krabbe, (Elliott, et al., 2016) [1]. We also provide the mass spectrometry peak areas for the product and internal standard ions typically observed with a dried blood spot punch from a random newborn, and we provide the daily variation of the daily mean activities for all 6 enzymes.

Pilot study of newborn screening for six lysosomal storage diseases using Tandem Mass Spectrometry.

BACKGROUND: There is current expansion of newborn screening (NBS) programs to include lysosomal storage disorders because of the availability of treatments that produce an optimal clinical outcome when started early in life. OBJECTIVE: To evaluate the performance of a multiplex-tandem mass spectrometry (MS/MS) enzymatic activity assay of 6 lysosomal enzymes in a NBS laboratory for the identification of newborns at risk for developing Pompe, Mucopolysaccharidosis-I (MPS-I), Fabry, Gaucher, Niemann Pick-A/B, and Krabbe diseases. METHODS AND RESULTS: Enzyme activities (acid α-glucosidase (GAA), galactocerebrosidase (GALC), glucocerebrosidase (GBA), α-galactosidase A (GLA), α-iduronidase (IDUA) and sphingomyeline phosphodiesterase-1 (SMPD-1)) were measured on ~43,000 de-identified dried blood spot (DBS) punches, and screen positive samples were submitted for DNA sequencing to obtain genotype confirmation of disease risk. The 6-plex assay was efficiently performed in the Washington state NBS laboratory by a single laboratory technician at the bench using a single MS/MS instrument. The number of screen positive samples per 100,000 newborns were as follows: GAA (4.5), IDUA (13.6), GLA (18.2), SMPD1 (11.4), GBA (6.8), and GALC (25.0). DISCUSSION: A 6-plex MS/MS assay for 6 lysosomal enzymes can be successfully performed in a NBS laboratory. The analytical ranges (enzyme-dependent assay response for the quality control HIGH sample divided by that for all enzyme-independent processes) for the 6-enzymes with the MS/MS is 5- to 15-fold higher than comparable fluorimetric assays using 4-methylumbelliferyl substrates. The rate of screen positive detection is consistently lower for the MS/MS assay compared to the fluorimetric assay using a digital microfluidics platform.

The effect of fixed charge modifications on electron capture dissociation.

Electron capture dissociation (ECD) studies of two modified amyloid beta peptides (20-29 and 25-35) were performed to investigate the role of H* radicals in the ECD of peptide ions and the free-radical cascade (FRC) mechanism. 2,4,6-Trimethylpyridinium (TMP) was used as the fixed charge tag, which is postulated to both trap the originally formed radical upon electron capture and inhibit the H* generation. It was found that both the number and locations of the fixed charge groups influenced the backbone and side-chain cleavages of these peptides in ECD. In general, the frequency and extent of backbone cleavages decreased and those of side-chain cleavages increased with the addition of fixed charge tags. A singly labeled peptide with the tag group farther away from the protonated site experienced a smaller abundance decrease in backbone cleavage fragments than the one with the tag group closer to the protonated site. Despite the nonprotonated nature of all charge carriers in doubly labeled peptide ions, several c and z* ions were still observed in their ECD spectra. Thus, although H* transfer may be important for the NC(alpha) bond cleavage, there also exist other pathways, which would require a radical migration via H* abstraction through space or via an amide superbase mechanism. Finally, internal fragment ions were observed in the ECD of these linear peptides, indicating that the important role of the FRC in backbone cleavages is not limited to the ECD of cyclic peptides.

Use of 18O labels to monitor deamidation during protein and peptide sample processing.

Nonenzymatic deamidation of asparagine residues in proteins generates aspartyl (Asp) and isoaspartyl (isoAsp) residues via a succinimide intermediate in a neutral or basic environment. Electron capture dissociation (ECD) can differentiate and quantify the relative abundance of these isomeric products in the deamidated proteins. This method requires the proteins to be digested, usually by trypsin, into peptides that are amenable to ECD. ECD of these peptides can produce diagnostic ions for each isomer; the c. + 58 and z - 57 fragment ions for the isoAsp residue and the fragment ion ((M + nH)((n-1)+.) - 60) corresponding to the side-chain loss from the Asp residue. However, deamidation can also occur as an artifact during sample preparation, particularly when using typical tryptic digestion protocols. With 18O labeling, it is possible to differentiate deamidation occurring during trypsin digestion which causes a +3 Da (18O1 + 1D) mass shift from the pre-existing deamidation, which leads to a +1-Da mass shift. This paper demonstrates the use of (18)O labeling to monitor three rapidly deamidating peptides released from proteins (calmodulin, ribonuclease A, and lysozyme) during the time course of trypsin digestion processes, and shows that the fast (approximately 4 h) trypsin digestion process generates no additional detectable peptide deamidations.

Probing the gas-phase folding kinetics of peptide ions by IR activated DR-ECD.

The effect of infrared (IR) irradiation on the electron capture dissociation (ECD) fragmentation pattern of peptide ions was investigated. IR heating increases the internal energy of the precursor ion, which often amplifies secondary fragmentation, resulting in the formation of w-type ions as well as other secondary fragments. Improved sequence coverage was observed with IR irradiation before ECD, likely due to the increased conformational heterogeneity upon IR heating, rather than faster breakdown of the initially formed product ion complex, as IR heating after ECD did not have similar effect. Although the ECD fragment ion yield of peptide ions does not typically increase with IR heating, in double resonance (DR) ECD experiments, fragment ion yield may be reduced by fast resonant ejection of the charge reduced molecular species, and becomes dependent on the folding state of the precursor ion. In this work, the fragment ion yield was monitored as a function of the delay between IR irradiation and the DR-ECD event to study the gas-phase folding kinetics of the peptide ions. Furthermore, the degree of intracomplex hydrogen transfer of the ECD fragment ion pair was used to probe the folding state of the precursor ion. Both methods gave similar refolding time constants of approximately 1.5 s(-1), revealing that gaseous peptide ions often refold in less than a second, much faster than their protein counterparts. It was also found from the IR-DR-ECD study that the intramolecular H. transfer rate can be an order of magnitude higher than that of the separation of the long-lived c/z product ion complexes, explaining the common observation of c. and z type ions in ECD experiments.

Quantitating the relative abundance of isoaspartyl residues in deamidated proteins by electron capture dissociation.

Relative quantitation of aspartyl and isoaspartyl residue mixtures from asparagine deamidation is demonstrated using electron capture dissociation without prior HPLC separation. The method utilizes the linear relationship found between the relative abundance of the isoaspartyl diagnostic ion, z(n)-57, and % isoaspartyl content based on the ECD spectra of known isoaspartyl/aspartyl mixtures of synthetic peptides. The observed linearity appears to be sequence independent because the relationship exists despite sequence variations and changes in backbone fragment abundances when isoaspartyl and aspartyl residues are interchanged. Furthermore, a new method to calculate the relative abundances of isomer from protein deamidation without synthetic peptides is proposed and tested using a linear peptide released by protein digestion that contains the deamidation site. The proteolytic peptide can be rapidly aged to the expected 3:1 (isoaspartyl:aspartyl) mixture to generate a two-point calibration standard for ECD analysis. The procedure can then be used to determine the relative abundance of deamidation products from in vivo or in vitro protein aging experiments.

The effect of radical trap moieties on electron capture dissociation spectra of substance P.

To further test the hypothesis that electron capture dissociation (ECD) involves long-lived radical intermediates and radical migration occurs within these intermediates before fragmentation, radical trap moieties were attached to peptides with the assumption that they would reduce fragmentation by decreasing the mobility of the radical. Coumarin labels were chosen for the radical traps, and unlabeled, singly-labeled, and doubly-labeled Substance P were analyzed by ECD. The results demonstrated a correlation between the number and position of tags on the peptide and the intensity of side-chain cleavages observed, as well as an inverse correlation between the number of tags on the peptide and the intensity of backbone cleavages. Addition of radical traps to the peptide inhibits backbone cleavages, suggesting that either radical mobility is required for these cleavages, or new noncovalent interactions prevent separation of backbone cleavage fragments. The enhancement of side-chain cleavages and the observation of new side-chain cleavages associated with aromatic groups suggest that the gas-phase conformation of this peptide is substantially distorted from untagged Substance P and involves previously unobserved interactions between the coumarin tags and the phenylalanine residues. Furthermore, the use of a double resonance (DR)-ECD experiment showed that these side-chain losses are all products of long-lived radical intermediate species, which suggests that steric hindrance prevents the coumarin-localized radical from interacting with the backbone while simultaneously increasing the radical rearrangements with the side chains.

Use of a double resonance electron capture dissociation experiment to probe fragment intermediate lifetimes.

The relative abundances of fragment ions in electron capture dissociation (ECD) are often greatly affected by the secondary and tertiary structures of the precursor ion, and have been used to derive the gas-phase conformations of the protein ions. In this study, it is found that resonance ejection of the charge reduced molecular ion during ECD resulted in significant changes in many fragment ion populations. The ratio of the ion peak intensities in the double resonance (DR)-ECD to that in the normal ECD experiment can be used to calculate the lifetime of the radical intermediates from which these fragments are derived. These lifetimes are often in the ms range, a time sufficiently long to facilitate multiple free radical rearrangements. These ratios correlate with the intramolecular noncovalent interactions in the precursor ion, and can be used to deduce information about the gas-phase conformation of peptide ions. DR-ECD experiments can also provide valuable information on ECD mechanisms, such as the importance of secondary electron capture and the origin of c./z ions.

Long-lived electron capture dissociation product ions experience radical migration via hydrogen abstraction.

To explore the mechanism of electron capture dissociation (ECD) of linear peptides, a set of 16-mer peptides were synthesized with deuterium labeled on the alpha-carbon position of four glycines. The ECD spectra of these peptides showed that such peptides exhibit a preference for the radical to migrate to the alpha-carbon position on glycine via hydrogen (or deuterium) abstraction before the final cleavage and generation of the detected product ions. The data show c-type fragment ions, ions corresponding to the radical cation of the c-type fragments, c*, and they also show c*-1 peaks in the deuterated peptides only. The presence of the c*-1 peaks is best explained by radical-mediated scrambling of the deuterium atoms in the long-lived, metastable, radical intermediate complex formed by initial electron capture, followed by dissociation of the complex. These data suggest the presence of at least two mechanisms, one slow, one fast. The abundance of H* and -CO losses from the precursor ion changed upon deuterium labeling indicating the presence of a kinetic isotope effect, which suggests that the values reported here represent an underestimation of radical migration and H/D scrambling in the observed fragments.

Detecting deamidation products in proteins by electron capture dissociation.

A nonenzymatic posttranslational modification of proteins and peptides is the spontaneous deamidation of asparaginyl residues via a succinimide intermediate to form a varying mixture of aspartyl and isoaspartyl residues. The isoaspartyl residue is generally difficult to detect particularly using mass spectrometry because isoaspartic acid is isomeric with aspartic acid so that there is no mass difference. However, electron capture dissociation has demonstrated the ability to differentiate the two isoforms in synthetic peptides using unique diagnostic ions for each form; the cr. + 58 and z(l-r) - 57 fragment ions for the isoAsp form and the Asp side chain loss ((M + nH)(n-1)+. - 60) for the Asp form. Shown here are three examples of isoaspartyl detection in peptides from proteins; a deamidated tryptic peptide of cytochrome c, a tryptic peptide from unfolded and deamidated ribonuclease A, and a tryptic peptide from calmodulin deamidated in its native state. In all cases, the cr. + 58 and z(l-r) - 57 ions allowed the detection and localization of isoaspartyl residues to positions previously occupied by asparaginyl residues. The (M + nH)(n-1)+. - 60 ions were also detected, indicating the presence of aspartyl residues. Observation of these diagnostic ions in peptides from proteins shows that the method is applicable to defining the isomerization state of deamidated proteins.

Differentiation of aspartic and isoaspartic acids using electron transfer dissociation.

Electron-transfer dissociation allows differentiation of isoaspartic acid and aspartic acid residues using the same c + 57 and z - 57 peaks that were previously observed with electron capture dissociation. These peaks clearly define both the presence and the position of isoaspartic acid residues and they are relatively abundant. The lower resolution of the ion trap instrument makes detection of the aspartic acid residue's diagnostic peak difficult because of interference with side-chain fragment ions from arginine residues, but the aspartic acid residues are still clearly observed in the backbone cleavages and can be inferred from the absence of the isoaspartic acid diagnostic ions.

Deamidation: Differentiation of aspartyl from isoaspartyl products in peptides by electron capture dissociation.

Deamidation of asparaginyl and isomerization of aspartyl residues in proteins proceed through a succinimide intermediate producing a mixture of aspartyl and isoaspartyl residues. Isoaspartic acid is an isomer of aspartic acid with the C(beta) incorporated into the backbone, thus increasing the length of the protein backbone by one methylene unit. This post-translation modification is suspected to contribute to the aging of proteins and to protein folding disorders such as Alzheimer's disease, so that differentiating the two isomers becomes important. This manuscript reports that distinguishing aspartyl from isoaspartyl residues in peptides has been accomplished by electron capture dissociation (ECD) using a Fourier transform mass spectrometer (FTMS). Model peptides with aspartyl residues and their isoaspartyl analogs were examined and unique peaks corresponding to c(n)*+58 and z(l-n)-57 fragment ions (n, position of Asp; l, total number of amino acids in the peptide) were found only in the spectra of the peptides with isoaspartyl residues. The proposed fragmentation mechanism involves cleavage of the C(alpha)-C(beta) backbone bond, therefore splitting the isoaspartyl residue between the two fragments. Also, a complementary feature observed specific to aspartyl residues was the neutral loss of the aspartic acid side chain from the charge reduced species. CAD spectra of the peptides from the same instrument demonstrated the improved method because previously published CAD methods rely on the comparison to the spectra of standards with aspartyl residues. The potential use of the top-down approach to detect and resolve products from the deamidation of asparaginyl and isomerization of aspartyl residues is discussed.

Color test for the detection of resin-bound aldehyde in solid-phase combinatorial synthesis.

We report the development of a sensitive and specific color test for the detection of the presence of resin-bound aldehyde groups using 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole (Purpald). Aldehyde resin turns dark-brown to purple after a 5 min reaction followed by a 10 min air oxidation period. Resins that possess other functional groups (i.e., ketone, ester, amide, alcohol, and carboxylic acid) do not change color under the same conditions. The detection limit is 20 micromol/g for polystyrene-based aldehyde resins.