RESUMO
Measurement of methylmalonic acid (MMA) plays an important role in the diagnosis of vitamin B12 deficiency. Vitamin B12 is an essential cofactor for the enzymatic carbon rearrangement of methylmalonyl-CoA (MMA-CoA) to succinyl-CoA (SA-CoA), and the lack of vitamin B12 leads to elevated concentrations of MMA. Measurement of MMA in biological samples is complicated because of the presence of succinic acid (SA), isomer of MMA. We developed a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for MMA. The method utilizes derivatization and positive ion mode ionization, which is specific to polycarboxylic acids (MMA and SA are dicarboxylic acids), while derivatives of monocarboxylic acids at these conditions are not ionizable and not detectable. The only organic acid, other than MMA, that is detected in this method is SA. The described method does not require chromatographic resolution of the peaks of MMA and SA; quantitative measurement of MMA is performed using a deconvolution algorithm, which mathematically resolves signal corresponding to MMA, from the combined signal of MMA/SA. Because of the high selectivity of detection, this method utilizes isocratic chromatographic separation; reconditioning and re-equilibration of the chromatographic column between injections is unnecessary. The above features allow high-throughput analysis of MMA with injection-to-injection cycle time of approximately 1 minute.
Assuntos
Ácido Metilmalônico , Espectrometria de Massas em Tandem , Carbono , Cromatografia Líquida/métodos , Coenzima A , Ácido Metilmalônico/química , Succinatos , Espectrometria de Massas em Tandem/métodos , VitaminasRESUMO
N-terminal sequence of parathyroid hormone-related protein (PTHrP) has close homology to parathyroid hormone (PTH). In health, both PTH and PTHrP participate in calcium regulation and homeostasis, but some of the functions, such as regulation of bone development, teeth eruption, calcium regulation in central nervous system, and calcium regulation during pregnancy and fetal development, are unique to PTHrP. In pathology, PTHrP is involved in activation of the pathways, allowing tumor cells to form bone metastasis. In contemporary clinical practice, measurements of PTHrP are used for diagnosing and management of patients suspected of hypercalcemia of malignancy. We describe high-sensitivity, high-specificity LC-MS/MS method for measurement of PTHrP. Sample preparation in this method is performed as follows: internal standard (15N labeled PTHrP) is added to plasma samples. PTHrP and the internal standard are enriched from the samples using anti-PTHrP antibody conjugated to magnetic beads. The beads are washed, PTHrP is digested with trypsin, and a PTHrP-specific signature peptide is analyzed using LC-MS/MS. The lower limit of detection, limit of quantitation, and upper limit of linearity of the assay are 0.5, 2, and 600 pmol/L; total imprecision of the method is <10%. Reference intervals for PTHrP established using this method in samples of healthy women and men are <3.4 pmol/L and < 2.3 pmol/L, respectively. The method has acceptable performance for use in clinical diagnostic applications.
Assuntos
Neoplasias Ósseas , Proteína Relacionada ao Hormônio Paratireóideo , Cálcio , Cromatografia Líquida , Feminino , Humanos , Masculino , Hormônio Paratireóideo/metabolismo , Espectrometria de Massas em Tandem , TripsinaRESUMO
Although reproductive factors have been repeatedly associated with lung cancer risk, no study to date has directly evaluated the relationship with endogenous sex hormones nor with aromatase activity in postmenopausal never-smoking women. A case-control study of 397 incident lung cancer cases and their individually matched controls, nested within the Shanghai Women's Health Study, was conducted among postmenopausal women who were lifetime never smokers. Prediagnostic concentrations of sex hormones was quantitated using LC-MS/MS assays in plasma. The product-substrate molar ratio of estrone to androstenedione was used as an index of aromatase activity (IAA). Multivariable conditional logistic regression models were used to calculate odds ratios (ORs) for lung cancer. Baseline concentrations of estradiol, free testosterone and IAA were inversely associated with subsequent risk of lung cancer in multivariable-adjusted models. When further adjusted for body mass index, the inverse association with estradiol was attenuated and no longer statistically significant, but the association with free testosterone and IAA remained. In analyses confined to participants having never used menopausal hormone therapy in 376 case-control pairs, the inverse association with free testosterone and IAA was slightly strengthened. OR for the highest vs the lowest quartile of free testosterone was 0.55 (95% CI = 0.34-0.90; Ptrend = .03), and the corresponding OR for IAA was 0.57 (95% CI = 0.34-0.96; Ptrend = .04). Our study, for the first time, suggests that higher levels of circulating free testosterone and estimated aromatase activity may be associated with lower lung cancer risk in postmenopausal never-smoking women.
Assuntos
Neoplasias Pulmonares , Globulina de Ligação a Hormônio Sexual , Aromatase , Estudos de Casos e Controles , China/epidemiologia , Cromatografia Líquida , Estradiol , Feminino , Hormônios Esteroides Gonadais , Humanos , Modelos Logísticos , Neoplasias Pulmonares/epidemiologia , Neoplasias Pulmonares/etiologia , Pós-Menopausa , Estudos Prospectivos , Fatores de Risco , Fumar/efeitos adversos , Espectrometria de Massas em Tandem , TestosteronaRESUMO
Isotopic information determined by mass spectrometry can be used in a wide variety of applications. Broadly speaking these could be classified as "passive" applications, meaning that they use naturally occurring isotopic information, and "active" applications, meaning that the isotopic distributions are manipulated in some way. The classic passive application is the determination of chemical composition by comparing observed isotopic patterns of molecules to theoretically calculated isotopic patterns. Active applications include isotope exchange experiments of a variety of types, as well as isotope labeling in tracing studies and to provide references for quantitation. Regardless of the type of application considered, the problem of theoretical calculation of isotopic patterns almost invariably arises. This chapter reviews a number of application examples and computational approaches for isotopic studies in mass spectrometry.
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Algoritmos , Isótopos/análise , Espectrometria de Massas/métodos , Biologia Computacional , Marcação por IsótopoRESUMO
BACKGROUND: Hemoglobinopathies and thalassemias are the most common genetically determined disorders. Current screening methods include cation-exchange HPLC and electrophoresis, the results of which can be ambiguous because of limited resolving power. Subsequently, laborious genetic testing is required for confirmation. METHODS: We performed a top-down tandem mass spectrometry (MS/MS) approach with a fast data acquisition (3 min), ultrahigh mass accuracy, and extensive residue cleavage by use of positive electrospray ionization 21 Tesla Fourier transform ion cyclotron resonance-tandem mass spectrometry (21 T FT-ICR MS/MS) for hemoglobin (Hb) variant de novo sequencing and ß-thalassemia diagnosis. RESULTS: We correctly identified all Hb variants in blind analysis of 18 samples, including the first characterization of homozygous Hb Himeji variant. In addition, an Hb heterozygous variant with isotopologue mass spacing as small as 0.0194 Da (Hb AD) was resolved in both precursor ion mass spectrum (MS1) and product ion mass spectrum (MS2). In blind analysis, we also observed that the abundance ratio between intact δ and ß subunits (δ/ß) or the abundance ratio between intact δ and α subunits (δ/α) could serve to diagnose ß-thalassemia trait caused by a mutation in 1 HBB gene. CONCLUSIONS: We found that 21 T FT-ICR MS/MS provides a benchmark for top-down MS/MS analysis of blood Hb. The present method has the potential to be translated to lower resolving power mass spectrometers (lower field FT-ICR mass spectrometry and Orbitrap) for Hb variant analysis (by MS1 and MS2) and ß-thalassemia diagnosis (MS1).
Assuntos
Análise de Fourier , Hemoglobinopatias/sangue , Hemoglobinas/química , Espectrometria de Massas/métodos , Espectrometria de Massas em Tandem/métodos , Talassemia beta/sangue , Sequência de Aminoácidos , Ciclotrons , Variação Genética , Hemoglobinopatias/genética , Humanos , Sensibilidade e Especificidade , Análise de Sequência de Proteína/métodos , alfa-Globinas/química , Globinas beta/química , Talassemia beta/genética , Globinas delta/químicaRESUMO
Leflunomide is a prodrug that is metabolized to the active metabolite, teriflunomide (A77 1726), to inhibit the enzyme dihydroorotate dehydrogenase and decrease the synthesis of pyrimidine nucleotides for DNA and RNA synthesis. Teriflunomide is primarily used for the treatment of rheumatoid arthritis and multiple sclerosis.A liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed and validated to quantify the drug teriflunomide over a concentration range of 5 ng/mL-200 µg/mL in serum or plasma. The calibration curve was divided into two separate overlapping regions of the analytical measurement range, with a high curve and a low curve range. Samples are first analyzed using the high-range calibration curve after a 100-fold dilution of the sample extract. Samples falling below the upper curve region are evaluated again without dilution and quantified, if possible, against the low curve calibration standards. This method can be used to support therapeutic drug monitoring of patients that are administered with leflunomide therapy.
Assuntos
Cromatografia Líquida , Crotonatos/farmacocinética , Imunossupressores/farmacocinética , Leflunomida/farmacocinética , Espectrometria de Massas em Tandem , Toluidinas/farmacocinética , Crotonatos/química , Humanos , Hidroxibutiratos , Leflunomida/química , Estrutura Molecular , Nitrilas , Toluidinas/químicaRESUMO
BACKGROUND: Parathyroid hormone-related protein (PTHrP) is involved in intracellular calcium regulation, neural cell proliferation and synaptic transmission. To date, no studies have been performed to evaluate the potential of PTHrP concentrations in cerebrospinal fluid (CSF) as a biomarker of brain pathophysiology. In this study we evaluated the association between CSF concentrations of PTHrP with the core CSF biomarkers of Alzheimer's disease (AD). METHODS: PTHrP and calcium were analysed using validated mass spectrometry based methods in a set of CSF samples that tested positive (nâ¯=â¯45) and negative (nâ¯=â¯45) for the AD biomarkers, including total tau protein (T-tau), phosphorylated tau protein (P-tau) and amyloid-ß 42 (Aß42). The measured CSF concentrations of PTHrP and calcium (Ca) were evaluated for association with AD CSF biomarkers. RESULTS: PTHrP and Ca concentrations in CSF samples ranged between 25 and 137â¯pmol/L and 0.92-1.53â¯mmol/L, respectively. Higher concentrations of PTHrP were observed in association with increased concentrations of T-tau and P-tau in the AD and the control group; while a stronger correlation was observed in the control group (ρâ¯=â¯0.6, pâ¯<â¯0.0001; and ρâ¯=â¯0.72, pâ¯<â¯0.0001, for T-tau and P-tau, respectively). Negative correlation was observed between concentrations of PTHrP and Aß42 in the AD group (ρâ¯=â¯0.27, pâ¯=â¯0.015). A statistically significantly lower ratio Aß42/PTHrP was observed in the AD group (pâ¯<â¯0.0001). CONCLUSION: In the current study, we observed an association of PTHrP concentrations with concentrations of clinically used CSF biomarkers of AD. Concentrations of PTHrP were positively correlated with concentrations of T-tau and P-tau, suggesting an association with neuronal secretion and function, which is reduced upon progression to AD pathology. Our data suggest potential utility of the Aß42/PTHrP ratio in assessment of AD progression.
RESUMO
Harmonization of diagnostic test results is fundamental to the effective use of laboratory testing in the diagnosis, treatment, and monitoring of disease. Formal approaches to harmonization and standardization provide a rigorous and high-quality roadmap to this end, although the formal harmonization process can be long and complex. In the meantime, more informal approaches to harmonization can provide a useful pathway to improved harmonization in the short term. Factors relevant to harmonization are discussed with particular attention to protein assays using LC-MS/MS. Published formal and informal harmonization projects are provided as examples, including lessons drawn from these projects.
Assuntos
Cromatografia Líquida , Proteínas/análise , Espectrometria de Massas em Tandem , Cromatografia Líquida/métodos , Cromatografia Líquida/normas , Humanos , Padrões de Referência , Espectrometria de Massas em Tandem/métodos , Espectrometria de Massas em Tandem/normasRESUMO
MOTIVATION: Using mass spectrometry to measure the concentration and turnover of the individual proteins in a proteome, enables the calculation of individual synthesis and degradation rates for each protein. Software to analyze concentration is readily available, but software to analyze turnover is lacking. Data analysis workflows typically don't access the full breadth of information about instrument precision and accuracy that is present in each peptide isotopic envelope measurement. This method utilizes both isotope distribution and changes in neutromer spacing, which benefits the analysis of both concentration and turnover. RESULTS: We have developed a data analysis tool, DeuteRater, to measure protein turnover from metabolic D 2 O labeling. DeuteRater uses theoretical predictions for label-dependent change in isotope abundance and inter-peak (neutromer) spacing within the isotope envelope to calculate protein turnover rate. We have also used these metrics to evaluate the accuracy and precision of peptide measurements and thereby determined the optimal data acquisition parameters of different instruments, as well as the effect of data processing steps. We show that these combined measurements can be used to remove noise and increase confidence in the protein turnover measurement for each protein. AVAILABILITY AND IMPLEMENTATION: Source code and ReadMe for Python 2 and 3 versions of DeuteRater are available at https://github.com/JC-Price/DeuteRater . Data is at https://chorusproject.org/pages/index.html project number 1147. Critical Intermediate calculation files provided as Tables S3 and S4. Software has only been tested on Windows machines. CONTACT: jcprice@chem.byu.edu. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Regulação da Expressão Gênica , Espectrometria de Massas/métodos , Peptídeos/análise , Proteoma/genética , Proteômica/métodos , Software , Animais , Isótopos , Cinética , Camundongos , Peptídeos/genética , Peptídeos/metabolismo , Proteoma/metabolismoRESUMO
HLA-DM and class II associated invariant chain (Ii) are key cofactors in the MHC class II (MHCII) antigen processing pathway. We used tandem mass spectrometry sequencing to directly interrogate the global impact of DM and Ii on the repertoire of MHCII-bound peptides in human embryonic kidney 293T cells expressing HLA-DQ molecules in the absence or presence of these cofactors. We found that Ii and DM have a major impact on the repertoire of peptides presented by DQ1 and DQ6, with the caveat that this technology is not quantitative. The peptide repertoires of type 1 diabetes (T1D) associated DQ8, DQ2, and DQ8/2 are altered to a lesser degree by DM expression, and these molecules share overlapping features in their peptide binding motifs that are distinct from control DQ1 and DQ6 molecules. Peptides were categorized into DM-resistant, DM-dependent, or DM-sensitive groups based on the mass spectrometry data, and representative peptides were tested in competitive binding assays and peptide dissociation rate experiments with soluble DQ6. Our data support the conclusion that high intrinsic stability of DQ-peptide complexes is necessary but not sufficient to confer resistance to DM editing, and provide candidate parameters that may be useful in predicting the sensitivity of T-cell epitopes to DM editing.
Assuntos
Diabetes Mellitus Tipo 1/imunologia , Antígenos HLA-D/metabolismo , Antígenos HLA-DQ/metabolismo , Motivos de Aminoácidos/genética , Apresentação de Antígeno , Antígenos/metabolismo , Antígenos de Diferenciação de Linfócitos B/metabolismo , Simulação por Computador , Epitopos de Linfócito T/genética , Células HEK293 , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Peptídeos/metabolismo , Ligação Proteica , Estabilidade Proteica , Espectrometria de Massas em TandemRESUMO
To improve efficiency in our mass spectrometry laboratories we have made efforts to reduce the number of calibration standards utilized for quantitation over time. We often analyze three or more batches of 96 samples per day, on a single instrument, for a number of assays. With a conventional calibration scheme at six concentration levels this amounts to more than 5000 calibration points per year. Modern LC-tandem mass spectrometric instrumentation is extremely rugged however, and isotopically labelled internal standards are widely available. This made us consider whether alternative calibration strategies could be utilized to reduce the number of calibration standards analyzed while still retaining high precision and accurate quantitation. Here we demonstrate how, by utilizing a single calibration point in each sample batch, and using the resulting response factor (RF) to update an existing, historical response factor (HRF), we are able to obtain improved precision over a conventional multipoint calibration approach, as judged by quality control samples. The laboratory component of this study was conducted with an existing LC tandem mass spectrometric method for three androgen analytes in our production laboratory. Using examples from both simulated and laboratory data we illustrate several aspects of our single point alternative calibration strategy and compare it with a conventional, multipoint calibration approach. We conclude that both the cost and burden of preparing multiple calibration standards with every batch of samples can be reduced while at the same time maintaining, or even improving, analytical quality.
Assuntos
Cromatografia Líquida/normas , Controle de Qualidade , Espectrometria de Massas em Tandem/normas , CalibragemRESUMO
BACKGROUND: For many years, basic and clinical researchers have taken advantage of the analytical sensitivity and specificity afforded by mass spectrometry in the measurement of proteins. Clinical laboratories are now beginning to deploy these work flows as well. For assays that use proteolysis to generate peptides for protein quantification and characterization, synthetic stable isotope-labeled internal standard peptides are of central importance. No general recommendations are currently available surrounding the use of peptides in protein mass spectrometric assays. CONTENT: The Clinical Proteomic Tumor Analysis Consortium of the National Cancer Institute has collaborated with clinical laboratorians, peptide manufacturers, metrologists, representatives of the pharmaceutical industry, and other professionals to develop a consensus set of recommendations for peptide procurement, characterization, storage, and handling, as well as approaches to the interpretation of the data generated by mass spectrometric protein assays. Additionally, the importance of carefully characterized reference materials-in particular, peptide standards for the improved concordance of amino acid analysis methods across the industry-is highlighted. The alignment of practices around the use of peptides and the transparency of sample preparation protocols should allow for the harmonization of peptide and protein quantification in research and clinical care.
Assuntos
Técnicas de Laboratório Clínico , Espectrometria de Massas , Peptídeos/análise , Proteômica , Manejo de Espécimes , Guias como Assunto , Humanos , Peptídeos/isolamento & purificação , PesquisadoresRESUMO
Steroid concentrations in stimulated follicular fluid (sFF) samples have been linked to the quality of oocytes used in IVF treatments. Most of the published studies focused on evaluating the association of the IVF outcomes with only a few of the steroids, measured by immunoassays (IA). We performed a treatment outcome, prospective cohort study using stimulated FF sampled from 14 infertile women undergoing IVF treatment; single oocyte was used per IVF cycle. Fourteen endogenous steroids were analyzed in 22 ovarian follicle aspirations, which corresponded to the embryos used in the IVF. Ten oocytes were associated with live birth (LB) and 12 with no pregnancy (NP). Steroids were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods. Differences in distribution of concentrations in association with the pregnancy outcome (LB or NP), and receiver operating characteristic (ROC) curves analysis were performed for the entire cohort and for within-women data. The predominant androgen and estrogen in stimulated sFF were androstenedione (A4) and estradiol (E2), respectively. Lower concentrations of pregnenolone (Pr), lower ratios of A4/ dehydroepiandrosterone (DHEA), testosterone (Te)/DHEA, and greater ratios of E2/Te, and estrone/A4 were observed in sFF samples associated with LB. Among the oocytes associated with NP, in four out of 12 samples total concentration of androgens was above the distribution of the concentrations in the oocytes corresponding to the LB group. Observations of the study indicated increased consumption of precursors and increased biosynthesis of estrogens in the follicles associated with LB. Our data suggest that potentially steroid profiles in sFF obtained during oocyte retrieval may serve as biomarkers for selection of the best embryo to transfer after IVF.
Assuntos
Fertilização in vitro , Líquido Folicular/química , Indução da Ovulação , Esteroides/análise , Adulto , Androstenodiona/análise , Desidroepiandrosterona/análise , Estradiol/análise , Estrona/análise , Feminino , Humanos , Infertilidade Feminina , Nascido Vivo , Oócitos/química , Oócitos/citologia , Gravidez , Pregnenolona/análise , Estudos Prospectivos , Espectrometria de Massas em Tandem , Testosterona , Adulto JovemRESUMO
HLA-DM is essential for editing peptides bound to MHC class II, thus influencing the repertoire of peptides mediating selection and activation of CD4(+) T cells. Individuals expressing HLA-DQ2 or DQ8, and DQ2/8 trans-dimers, have elevated risk for type 1 diabetes (T1D). Cells coexpressing DM with these DQ molecules were observed to express elevated levels of CLIP (Class II associated invariant chain peptide). Relative resistance to DM-mediated editing of CLIP was further confirmed by HPLC-MS/MS analysis of eluted peptides, which also demonstrated peptides from known T1D-associated autoantigens, including a shared epitope from ZnT8 that is presented by all four major T1D-susceptible DQ molecules. Assays with purified recombinant soluble proteins confirmed that DQ2-CLIP complexes are highly resistant to DM editing, whereas DQ8-CLIP is partially sensitive to DM, but with an apparent reduction in catalytic potency. DM sensitivity was enhanced in mutant DQ8 molecules with disruption of hydrogen bonds that stabilize DQ8 near the DM-binding region. Our findings show that T1D-susceptible DQ2 and DQ8 share significant resistance to DM editing, compared with control DQ molecules. The relative resistance of the T1D-susceptible DQ molecules to DM editing and preferential presentation of T1D-associated autoantigenic peptides may contribute to the pathogenesis of T1D.
Assuntos
Antígenos de Diferenciação de Linfócitos B/imunologia , Diabetes Mellitus Tipo 1/imunologia , Antígenos HLA-D/imunologia , Antígenos HLA-DQ/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Sequência de Aminoácidos , Autoantígenos/imunologia , Linfócitos T CD4-Positivos/imunologia , Linhagem Celular , Células HEK293 , Antígenos HLA-DQ/genética , Humanos , Ativação Linfocitária/imunologia , Dados de Sequência MolecularRESUMO
We describe a direct method of measurement of free estradiol using equilibrium dialysis followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Serum aliquots and internal standards are extracted by liquid-liquid extraction using methyl-tert-butyl ether (MTBE) followed by derivatization with dansyl chloride. An API 5500 mass spectrometer operated in positive electrospray mode is used for detection.
Assuntos
Análise Química do Sangue/métodos , Cromatografia Líquida/métodos , Estradiol/sangue , Espectrometria de Massas em Tandem/métodos , Estradiol/química , Estradiol/isolamento & purificação , Humanos , Extração Líquido-Líquido , Éteres Metílicos/químicaRESUMO
Measurement of methylmalonic acid (MMA) plays an important role in the diagnosis of vitamin B12 deficiency. Vitamin B12 is an essential cofactor for the enzymatic carbon rearrangement of methylmalonyl-CoA (MMA-CoA) to succinyl-CoA (SA-CoA), and the lack of vitamin B12 leads to elevated concentrations of MMA. Presence of succinic acid (SA) complicates the analysis because mass spectra of MMA and SA are indistinguishable, when analyzed in negative ion mode and the peaks are difficult to resolve chromatographically. We developed a method for the selective analysis of MMA that exploits the significant difference in fragmentation patterns of di-butyl derivatives of the isomers MMA and SA in a tandem mass spectrometer when analyzed in positive ion mode. Tandem mass spectra of di-butyl derivatives of MMA and SA are very distinct; this allows selective analysis of MMA in the presence of SA. The instrumental analysis is performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in positive ion mode, which is, in combination with selective extraction of acidic compounds, is highly selective for organic acids with multiple carboxyl groups (dicarboxylic, tricarboxylic, etc.). In this method organic acids with a single carboxyl group are virtually undetectable in the mass spectrometer; the only organic acid, other than MMA, that is detected by this method is its isomer, SA. Quantitative measurement of MMA in this method is performed using a deconvolution algorithm, which mathematically resolves the signal corresponding to MMA and does not require chromatographic resolution of the MMA and SA peaks. Because of its high selectivity, the method utilizes isocratic chromatographic separation; reconditioning and re-equilibration of the chromatographic column between injections is unnecessary. The above features of the method allow high-throughput analysis of MMA with analysis cycle time of 1 min.
Assuntos
Análise Química do Sangue/métodos , Cromatografia Líquida de Alta Pressão/métodos , Ácido Metilmalônico/sangue , Ácido Metilmalônico/urina , Espectrometria de Massas em Tandem/métodos , Urinálise/métodos , Humanos , Isomerismo , Limite de Detecção , Ácido Metilmalônico/química , Estatística como Assunto , Ácido Succínico/química , Fatores de TempoRESUMO
Aliquots of serum or plasma samples are combined with stable isotope labeled internal standard. Pancreatic polypeptide (PP) and its truncated variant PP3-36 are enriched by incubation with anti-PP antibody conjugated to magnetic beads. Peptides are eluted from beads in acidic buffer and the samples analyzed using liquid chromatography coupled with tandem mass spectrometry. Instrumental analysis of PP and PP3-36 is performed using electrospray ionization ESI in positive ion mode and multiple reaction monitoring (MRM) acquisition.