43 research outputs found

    MRCQuant- an accurate LC-MS relative isotopic quantification algorithm on TOF instruments

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    <p>Abstract</p> <p>Background</p> <p>Relative isotope abundance quantification, which can be used for peptide identification and differential peptide quantification, plays an important role in liquid chromatography-mass spectrometry (LC-MS)-based proteomics. However, several major issues exist in the relative isotopic quantification of peptides on time-of-flight (TOF) instruments: LC peak boundary detection, thermal noise suppression, interference removal and mass drift correction. We propose to use the Maximum Ratio Combining (MRC) method to extract MS signal templates for interference detection/removal and LC peak boundary detection. In our method, MRCQuant, MS templates are extracted directly from experimental values, and the mass drift in each LC-MS run is automatically captured and compensated. We compared the quantification accuracy of MRCQuant to that of another representative LC-MS quantification algorithm (msInspect) using datasets downloaded from a public data repository.</p> <p>Results</p> <p>MRCQuant showed significant improvement in the number of accurately quantified peptides.</p> <p>Conclusions</p> <p>MRCQuant effectively addresses major issues in the relative quantification of LC-MS-based proteomics data, and it provides improved performance in the quantification of low abundance peptides.</p

    Peptide Orientation Affects Selectivity in Ion-Exchange Chromatography

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    Here we demonstrate that separation of proteolytic peptides, having the same net charge and one basic residue, is affected by their specific orientation toward the stationary phase in ion-exchange chromatography. In electrostatic repulsion−hydrophilic interaction chromatography (ERLIC) with an anion-exchange material, the C-terminus of the peptides is, on average, oriented toward the stationary phase. In cation exchange, the average peptide orientation is the opposite. Data with synthetic peptides, serving as orientation probes, indicate that in tryptic/Lys-C peptides the C-terminal carboxyl group appears to be in a zwitterionic bond with the side chain of the C-terminal Lys/Arg residue. In effect, the side chain is then less basic than the N-terminus, accounting for the specific orientation of tryptic and Lys-C peptides. Analyses of larger sets of peptides, generated from lysates by either Lys-N, Lys-C, or trypsin, reveal that specific peptide orientation affects the ability of charged side chains, such as phosphate residues, to influence retention. Phosphorylated residues that are remote in the sequence from the binding site affect retention less than those that are closer. When a peptide contains multiple charged sites, then orientation is observed to be less rigid and retention tends to be governed by the peptide’s net charge rather than its sequence. These general observations could be of value in confirming a peptide’s identification and, in particular, phosphosite assignments in proteomics analyses. More generally, orientation accounts for the ability of chromatography to separate peptides of the same composition but different sequence

    Identification of a Blood-Based Protein Biomarker Panel for Lung Cancer Detection

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    Lung cancer is the deadliest cancer worldwide, mainly due to its advanced stage at the time of diagnosis. A non-invasive method for its early detection remains mandatory to improve patients’ survival. Plasma levels of 351 proteins were quantified by Liquid Chromatography-Parallel Reaction Monitoring (LC-PRM)-based mass spectrometry in 128 lung cancer patients and 93 healthy donors. Bootstrap sampling and least absolute shrinkage and selection operator (LASSO) penalization were used to find the best protein combination for outcome prediction. The PanelomiX platform was used to select the optimal biomarker thresholds. The panel was validated in 48 patients and 49 healthy volunteers. A 6-protein panel clearly distinguished lung cancer from healthy individuals. The panel displayed excellent performance: area under the receiver operating characteristic curve (AUC) = 0.999, positive predictive value (PPV) = 0.992, negative predictive value (NPV) = 0.989, specificity = 0.989 and sensitivity = 0.992. The panel detected lung cancer independently of the disease stage. The 6-protein panel and other sub-combinations displayed excellent results in the validation dataset. In conclusion, we identified a blood-based 6-protein panel as a diagnostic tool in lung cancer. Used as a routine test for high- and average-risk individuals, it may complement currently adopted techniques in lung cancer screening.publishedVersio

    Multiplexing Iduronate-2-Sulphatase (MPS-II) into a 7-Plex Lysosomal Storage Disorder MS/MS Assay Using Cold-Induced Phase Separation

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    Mucopolysaccharidosis type II (MPS-II, Hunter syndrome, OMIM:30990) is a lysosomal storage disorder (LSD) that results in iduronate 2-sulphatase (I2S) enzyme deficiency. MPS-II was added to the Recommended Uniform Screening Panel (RUSP) in August 2022; thus, there is an increased demand for multiplexing I2S into existing LSD screening assays. After incubation with LSD synthetic substrates, extracts are cleaned using liquid–liquid extraction with ethyl acetate or protein precipitation using acetonitrile (ACN). We investigated cold-induced water ACN phase separation (CIPS) to improve the combination of 6-plex and I2S extracts to create a 7-plex assay, and compared it to room temperature ACN and ethyl acetate liquid–liquid extraction. The extracts were dried and resuspended in the mobile phase, and then analyzed using an optimized 1.9 min injection-to-injection liquid chromatography method coupled with tandem mass spectrometry (LC-MS/MS). The combination of ACN and CIPS improved the detection for I2S products without significant detriment to other analytes, which is attributable to a more complete coagulation and separation of heme, proteins, and extracted residual salts. Using CIPS for sample cleanup in dried blood spots (DBS) appears to represent a promising and straightforward way of achieving cleaner sample extracts in a new 7-plex LSD screening panel
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