Lab Matters Fall 2024 | Page 91

APHL 2024 POSTER ABSTRACTS

This poster will cover new progress and challenges encountered as the program expands . Any exposure data generated upon launch will also be presented . This work will provide insight into the prenatal and perinatal screening model ’ s transferability , which will have implications beyond New Jersey . Further , the data generated will be shared with medical and public health leaders to inform screening policy decisions .

Presenter : Sarah Mustaqali , sarah . mustaqali @ doh . nj . gov

Empower the Quantitation of Amino Acids and Acylcarnitine for Inborn Error of Metabolism Using Flow Injection – Orbitrap Mass Spectrometer

J . Guo , Y . Song , K . Hassell , Thermo Fisher Scientific

Introduction : The screening of newborns for inborn error of metabolism ( IEM ) is considered the largest and most successful disease prevention system in the United States . The quantitation amino acids and acylcarnitines from dried blood spots ( DBS ) via flow injection analysis — triple quadrupole tandem mass spectrometry ( FIA-QqQ-MS / MS ) serves as the main first-tier screening method . Although the QqQ-MS provides great robustness and sensitivity , the assay is incapable of separating nominal mass isobaric compounds with similar transitions without liquid chromatography separation . The hybrid high-resolution accurate mass spectrometers ( HRAMS ), such as Orbitrap Exploris MS , can detect ions with higher levels of confidence due to the low ppm mass accuracy to distinguish isobaric interferences from matrices and good fragmentation spectra matching results against the spectra library

Method : We quantitated 12 amino acids and 13 acylcarnitines on a Thermo Scientific™ Vanquish™ UHPLC and a Thermo Scientific™ Orbitrap™ Exploris™ 120 mass spectrometer using the quality control dried blood spot ( DBS ) samples , reagents and consumables from ClinSpot ® LC-MS / MS Complete Kits , Amino Acids and Acylcarnitines in DBS – non-derivatized ( RECIPE ). The inter- and intraday precision measurements were performed on three different days with five times sample preparation per day . The quantitation was performed in the targeted-MS2 ( tMS2 ) scan and the data was analyzed in Thermo Scientific™ TraceFinder™ software . The full MS spectra over the m / z ranges of common metabolites were recorded for retrospective analysis of novel biomarker discovery .

Results : All the measured analyte values were within the target ranges listed by ClinChek – Control DBS specification sheet , except ornithine , which was slightly lower . This observation was consistent with the previous work on the verification of the same kit using a Vanquish MD HPLC and Thermo Scientific™ TSQ Quantis™ MD mass spectrometer , where a slight difference in ionization efficiency of the same analyte across different MS vendors was acknowledged . The inter- and intra-day precision measurements showed that for all analyte quantitation , the % RSD was below 12 % and the % CV of the IS was below 9 %, which showed that the developed method was highly robust and reproducible .

Conclusion : We developed a FIA-HRAMS / MS method to quantitate 12 amino acids and 13 acylcarnitines in a ClinSpot ® LC-MS / MS Complete Kits for Amino Acids and Acylcarnitines in DBS . The method showed good accuracy and precision measurements comparable to those from the TSQ Quantis MD MS using the same kit . In addition , Orbitrap MS provided high mass accuracy detection , full-MS spectra over the m / z ranges of common metabolites and complete MS2 spectra to increase the analyte identification confidence . The results highlighted that Orbitrap MS has the potential to improve detection accuracy and enable novel biomarker discovery in the IEM screening laboratories .

Presenter : Jingshu Guo , jingshu . guo @ thermofisher . com

Enhanced Detection of Hemoglobin Variants by Highresolution Accurate-mass ( HRAM ) Mass Spectrometry for Clinical Research

Y . Song , K . Hassell , E . Goucher , J . Guo , T . Correa , Thermo Fisher Scientific

Introduction : Hemoglobinopathies are one of the most prevalent genetic disorders , with more than 330,000 affected births occurring annually . Common techniques used in Hb analysis are electrophoretic and chromatographic assays . However , there are challenges to differentiate isomers ( i . e ., between Hb C and Hb E ), very similar masses ( i . e ., < 1 Da difference between normal beta and Hb C , Hb E and Hb D-Punjab ), or new variants due to their lack of resolution . Mass spectrometry gained popularity in clinical research because of its robustness and versatility in analyzing a wide range of samples and analytes from small molecules to proteins . Here , we present the top-down analysis for enhanced detection of various hemoglobin variants using Orbitrap Exploris mass spectrometry for clinical research .

Methods : Proteins were directly extracted from dried blood spots ( DBS ) and analyzed by Thermo Scientific™ Orbitrap Exploris™ 240 with BioPharma Option coupled to Vanquish HPLC system . A 3.2 mm disc was punched from the DBS cards to 96 well plate and 50 uL water was added to rehydrate . For protein precipitation , 150 uL acetonitrile was added and incubated at -20 º C for 15 minutes . After micro-centrifugation , 160 uL of supernatant was removed and 70 uL water was added to resuspend . For LC-MS analysis , 10 uL of supernatant was diluted eight times with mobile phase A and transferred to another 96-well plate . LC separation was performed on MAbPac RP column 4 um , 1 x 100 mm and data-dependent acquisition mode was operated .

Results : Proteins were directly extracted from dried blood spots using protein precipitation , allowing the entire sample preparation time to be less than one hour . Also , the LC-MS run time was less than 10 min per sample . The MAbPac column showed its capability of separating between different chains with minor overlap between normal beta and HbS beta chains . Data processing was performed by ProSightPD , resulting in ~ 45 % sequence coverage for both normal Hb and HbS beta chains . The mass difference between normal beta and Hb S beta chains is about 30 Da , which results in about 1.6 m / z difference at a charge state of 19 . This mass difference can often be hindered by other proteins or interferences from the matrix if the mass analyzer doesn ’ t have sufficient resolving power . Here we successfully applied Orbitrap resolution at 120,000 to isolate these similar masses for accurate variants or subunits identification including normal alpha , normal beta , Hb S and delta chains . The confirmation of the delta chain can be beneficial since its abundance is usually less than 0.5 % in adults , which shows potential for detecting low abundant variants using this method . Also , the calibration curve was generated to demonstrate the method ’ s feasibility which is not a typical quantitation method for hemoglobinopathies . For quantitation , three most abundant m / z values from two charge states were added to TraceFinder for