High Sensitivity Measurement of Parathyroid Hormone-Related Protein (PTHrP) in Plasma by LC-MS/MS.

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.

Endogenous sex hormones, aromatase activity and lung cancer risk in postmenopausal never-smoking women.

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.

DeuteRater: a tool for quantifying peptide isotope precision and kinetic proteomics.

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.

Peptidomic analysis of type 1 diabetes associated HLA-DQ molecules and the impact of HLA-DM on peptide repertoire editing.

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.

Recommendations for the Generation, Quantification, Storage, and Handling of Peptides Used for Mass Spectrometry-Based Assays.

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.

Mass spectrometry methods measured androgen and estrogen concentrations during pregnancy and in newborns of mothers with polycystic ovary syndrome.

OBJECTIVE: Little is known about the aetiology of polycystic ovary syndrome (PCOS). Some suggest that elevated maternal androgens during gestation play a causative role. This implies placental passage of androgens during pregnancy. The aim of this study is to compare androgen and estrogen concentrations in maternal serum during pregnancy and in umbilical cord blood, between mothers with PCOS and their offspring compared to controls. DESIGN: Prospective case-control study. METHODS: Maternal blood samples were collected around 20 weeks of gestation and at delivery. Umbilical cord blood was also taken at delivery. Androgens (testosterone (T), androstenedione (ADION), dehydroepiandrostenedione (DHEA)) and estrogens (estrone (E1), estradiol (E2), estriol (E3)) were measured using the liquid chromatography tandem mass spectrometry (LC-MS/MS) methods. RESULTS: At 20 weeks of gestation: T (P=0.019) and ADION (P=0.034) were higher in the PCOS mothers (pregnant with a girl), whereas DHEA, E1, E2, and E3 were not different. Maternal concentration at birth: T (P=0.004) and ADION (P=0.009) were also higher in the subgroup of PCOS mothers that were pregnant with a girl compared to the girl pregnancy controls. DHEA, E1, E2 and E3 were not different. In umbilical cord blood, no differences were found for T, ADION, DHEA, E2, E3, and AMH between the PCOS mothers and the controls respectively. E1 was lower in girls from PCOS mothers (P=0.007). CONCLUSIONS: Despite elevated maternal androgen concentrations during pregnancy in PCOS mothers, offspring showed no signs of elevated androgen concentrations in cord blood at birth using the latest highly specific LC-MS/MS methods.

LC-MS/MS Measurement of Parathyroid Hormone-Related Peptide.

INTRODUCTION: Parathyroid hormone-related peptide (PTHrP) is involved in activating pathways, allowing tumor cells to form bone metastases. Measurement of PTHrP is used for the diagnosis and clinical management of patients suspected of hypercalcemia of malignancy. We developed an LC-MS/MS method for measuring PTHrP, established sex-specific reference intervals, and assessed the method's performance. METHODS: PTHrP was enriched from plasma samples with rabbit polyclonal anti-PTHrP antibody conjugated to magnetic beads. Enriched PTHrP was digested with trypsin, and PTHrP-specific tryptic peptide was analyzed with 2-dimensional LC-MS/MS in multiple reaction monitoring mode. RESULTS: The lower limit of quantification was 0.6 pmol/L, and the upper limit of linearity was 600 pmol/L. Total imprecision was <10%. Very poor agreement was observed with the RIA (n = 207; Deming regression RIA = 0.059 × LC-MS/MS - 1.8, r = 0.483; Sy|x = 3.9). Evaluation of the clinical performance of the assay using samples from patients with and without hypercalcemia (n = 199) resulted in an area under the ROC curve of 0.874. In sets of consecutively analyzed routine samples of patients assessed for hypercalcemia, the PTHrP positivity rate by RIA (n = 1376) was 1.9%, and 26.6% by LC-MS/MS (n = 1705). Concentrations were below the lower limit of quantification in 95.6% of the samples by RIA and 2.0% by LC-MS/MS. CONCLUSIONS: PTHrP is a normal constituent in circulating blood and its concentrations are substantially underestimated by commercial RIAs, causing false-negative results in samples from patients suspected of hypercalcemia. Our observations suggest a link between increased concentrations of PTHrP in postmenopausal women with low body mass index and increased incidence of osteoporosis.

Direct Measurement of Free Estradiol in Human Serum and Plasma by Equilibrium Dialysis-Liquid Chromatography-Tandem Mass Spectrometry.

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.

High Sensitivity Measurement of Pancreatic Polypeptide and Its Variant in Serum and Plasma by LC-MS/MS.

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.

Exploratory study of the association of steroid profiles in stimulated ovarian follicular fluid with outcomes of IVF treatment.

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.

Mid-pregnancy, perinatal, and neonatal reproductive endocrinology: a prospective cohort study in twins and singleton control subjects.

OBJECTIVE: To answer the questions: Are perinatal reproductive hormone profiles different in case of a twin compared with a singleton pregnancy? Are reproductive endocrine profiles of twin girls influenced by their male co-twin and vice versa? DESIGN: Prospective cohort study from January 2004 to October 2009. SETTING: Not applicable. PATIENT(S): A total of 204 mothers of twins and 248 singleton control subjects, aged >18 years, pregnant with a twin or singleton and no endocrine disease or malignancy. INTERVENTION(S): Blood samples were collected at mid-gestation from the mother and at delivery from the mothers and the umbilical cords. Estrogens, androgens, sex hormone-binding globulin, progesterone, and gonadotropins were measured. MAIN OUTCOME MEASURE(S): Hormonal profiles were compared between singletons and twins, different types of twins, and opposite-sex and same-sex twins. RESULT(S): Estrogen and progesterone concentrations were higher in mothers of twins compared with singletons, but twin babies had lower estrogen and progesterone concentrations at birth. Opposite-sex twin girls did not have higher androgens in cord blood compared with same-sex twin girls. Boys of an opposite-sex twin had lower luteinizing hormone concentrations compared with dizygotic twin boys with a brother as a co-twin. CONCLUSION(S): Children from a twin are not overexposed to sex steroids at the time of birth, despite higher concentrations in their mothers, and girls from opposite sex twins do not show androgenic influences from their male co-twin. The female co-twin may influence the hypothalamic-pituitary-testicular axis of her brother via central inhibition.

Performance enhancement in the measurement of 5 endogenous steroids by LC-MS/MS combined with differential ion mobility spectrometry.

BACKGROUND: Challenges for steroid analysis by LC-MS/MS include low ionization efficiency, endogenous isobars with similar fragmentation patterns and chromatographic retention. Differential ion mobility spectrometry (DMS) provides an additional degree of separation prior to MS/MS detection, and shows promise in improving specificity of analysis. We developed a sensitive and specific method for measurement of corticosterone, 11-deoxycortisol, 11-deoxycorticosterone, 17-hydroxyprogesterone and progesterone in human serum and plasma using an ABSciex 5500 mass spectrometer equipped with a differential ion mobility interface. METHODS: 250µL aliquots of serum were spiked with deuterated internal standards and extracted with MTBE. The samples were analyzed using positive mode electrospray LC-DMS-MS/MS. The method was validated and compared with immunoassays and LC-MS/MS methods of reference laboratories. RESULTS: Inter and intra assay imprecision was <10%. Limits of quantification and detection in nmol/L were 0.18, 0.09 for corticosterone and 17-hydroxyprogesterone, 0.30, 0.16 for 11-deoxycortisol, 0.12, 0.06 for progesterone and 0.06, 0.03 for 11-deoxycorticosterone. Comparison for progesterone and 17-hydroxyprogesterone with immunoassay showed slopes of 0.97 and 1.0, intercepts of 0.16 and 0.10 and coefficients of determination (r(2)) of 0.92 and 0.97, respectively. Progesterone by immunoassay showed positive bias in samples measuring <3.18nmol/L. Reference intervals for progesterone and 11-deoxycorticosterone in post-menopausal women were found to be <2.88 and <0.28nmol/L respectively. CONCLUSIONS: We developed and validated an LC-DMS-MS/MS method for analysis of five endogenous steroids suitable for routine measurements in clinical diagnostic laboratories. Specificity gained with DMS allows reducing the complexity of sample preparation, decreasing LC run times and increasing speed of the analysis.

ERAAP and tapasin independently edit the amino and carboxyl termini of MHC class I peptides.

Effective CD8(+) T cell responses depend on presentation of a stable peptide repertoire by MHC class I (MHC I) molecules on the cell surface. The overall quality of peptide-MHC I complexes (pMHC I) is determined by poorly understood mechanisms that generate and load peptides with appropriate consensus motifs onto MHC I. In this article, we show that both tapasin (Tpn), a key component of the peptide loading complex, and the endoplasmic reticulum aminopeptidase associated with Ag processing (ERAAP) are quintessential editors of distinct structural features of the peptide repertoire. We carried out reciprocal immunization of wild-type mice with cells from Tpn- or ERAAP-deficient mice. Specificity analysis of T cell responses showed that absence of Tpn or ERAAP independently altered the peptide repertoire by causing loss as well as gain of new pMHC I. Changes in amino acid sequences of MHC-bound peptides revealed that ERAAP and Tpn, respectively, defined the characteristic amino and carboxy termini of canonical MHC I peptides. Thus, the optimal pMHC I repertoire is produced by two distinct peptide editing steps in the endoplasmic reticulum.

Dynamic thermal gradient gas chromatography.

The use of negative axial thermal gradients in gas chromatography (TGGC) has intrigued chromatographers since the early 1950s because of the dramatic narrowing of analyte bands and concomitant raised expectations for improving resolving power. However, technical difficulties experienced in construction of TGGC instrumentation and control of the temperature along the column have made its implementation and, hence, detailed study difficult. In this work, we describe a TGGC system capable of rapidly producing and varying thermal gradient profiles by simultaneous use of resistive heating and convective cooling. Heating and cooling rates as high as 1200 and 2500°C/min, respectively, allowed the creation of dynamic temperature gradients. The separation characteristics of TGGC with dynamically changing temperature gradients are demonstrated. A gradient velocity of 2.22cm/s provided repetitive separations every 45s, and injection band widths of 45s duration were transformed into approximately 1-s peak widths. Peak tailing for basic compounds was nearly eliminated. Dynamic TGGC allows unique control over separations, oftentimes improving resolution and detection signal-to-noise. Thermally controlled elution in TGGC holds great promise for performing smart separations in which the separation time window is most efficiently utilized, and optimized separations can be quickly achieved. Rapid adjustment of relative compound elution can be used to greatly reduce GC method development time.

Circulating estrone levels are associated prospectively with diabetes risk in men of the Framingham Heart Study.

OBJECTIVE: In postmenopausal women and preclinical murine models, estrogen administration reduces diabetes risk; however, the relationship of estradiol and estrone to diabetes in men is poorly understood. We determined the relationship between circulating estradiol and estrone levels and diabetes risk in community-dwelling men of the Framingham Heart Study (FHS). RESEARCH DESIGN AND METHODS: Cross-sectional relationships of estradiol and estrone levels with diabetes were assessed at examination 7 (1998-2001) in FHS generation 2 men (n = 1,458); prospective associations between hormone levels at examination 7 and incident diabetes were assessed 6.8 years later at examination 8. Type 2 diabetes mellitus was defined as fasting glucose >125 mg/dL, medication use, or both. Estradiol, estrone, and testosterone levels were measured with liquid chromatography-tandem mass spectrometry, and free estradiol and estrone were calculated. RESULTS: In cross-sectional models, men with elevated estrone and estradiol had 40% and 62% increased likelihoods of existing diabetes per cross-sectional doubling of estrone and estradiol levels, respectively. Free estrone (cross-sectional odds ratio 1.28 [95% CI 1.02-1.62], P = 0.04) was associated with impaired fasting glucose at examination 7. There was an increase in risk of existing diabetes with increasing quartiles of total and free estrone and estradiol and an increase in risk of incident diabetes with increasing quartiles of estrone levels. In multivariate longitudinal analyses, a twofold increase in total or free estrone levels at examination 7 was associated with 77 and 93% increases, respectively, in odds of incident diabetes at examination 8. CONCLUSIONS: Although both estradiol and estrone exhibit cross-sectional associations with diabetes in men, in longitudinal analyses estrone is a more sensitive marker of diabetes risk than is estradiol.

Correction for isotopic interferences between analyte and internal standard in quantitative mass spectrometry by a nonlinear calibration function.

Stable isotope-labeled internal standards are of great utility in providing accurate quantitation in mass spectrometry (MS). An implicit assumption has been that there is no "cross talk" between signals of the internal standard and the target analyte. In some cases, however, naturally occurring isotopes of the analyte do contribute to the signal of the internal standard. This phenomenon becomes more pronounced for isotopically rich compounds, such as those containing sulfur, chlorine, or bromine, higher molecular weight compounds, and those at high analyte/internal standard concentration ratio. This can create nonlinear calibration behavior that may bias quantitative results. Here, we propose the use of a nonlinear but more accurate fitting of data for these situations that incorporates one or two constants determined experimentally for each analyte/internal standard combination and an adjustable calibration parameter. This fitting provides more accurate quantitation in MS-based assays where contributions from analyte to stable labeled internal standard signal exist. It can also correct for the reverse situation where an analyte is present in the internal standard as an impurity. The practical utility of this approach is described, and by using experimental data, the approach is compared to alternative fits.

Measurement of thyroglobulin by liquid chromatography-tandem mass spectrometry in serum and plasma in the presence of antithyroglobulin autoantibodies.

BACKGROUND: Measurement of serum thyroglobulin (Tg) is used to monitor patients after treatment for differentiated thyroid carcinoma (TC). Difficulty in using Tg as a biomarker of the recurrence of TC in many patients stems from the presence of endogenous anti-Tg autoantibodies (Tg-AAbs), which can interfere with immunoassays (IAs) and cause false-negative results. METHODS: We enriched Tg from serum samples using rabbit polyclonal anti-Tg antiserum and protein precipitation. Unrelated proteins were partially depleted in the process. Enriched proteins were then denatured, reduced, and digested with trypsin after the addition of a winged internal standard peptide. A Tg-specific tryptic peptide was purified by immunoaffinity extraction and analyzed by 2-dimensional LC-MS/MS. Instrument cycle time was 6.5 min per sample. RESULTS: The lower limit of quantification was 0.5 ng/mL (0.76 fmol/mL dimer). Total imprecision of triplicate measurements in serum samples over 5 days was <10%. Comparison with a commercial IA using serum samples free of Tg-AAb (n = 73) showed Deming regression, IA = 1.00 * LC-MS/MS - 2.35, r = 0.982, standard error of the estimate (S(y|x)) = 9.52. In a set of Tg-AAb-positive samples that tested negative for Tg using IA (n = 71), concentrations determined by LC-MS/MS were ≥0.5 ng/mL in 23% of samples (median 1.2, range 0.7-11 ng/mL). CONCLUSIONS: The introduced method has acceptable performance characteristics for use in clinical diagnostic applications. The most substantial disagreement between methods was observed in Tg-AAb-positive samples with concentrations <2 ng/mL (determined with LC-MS/MS). The affinity-assisted enrichment strategy used for Tg in this method should be applicable to other biomarkers that have endogenous autoantibodies.

Regulation of HLA-DR peptide occupancy by histone deacetylase inhibitors.

Numerous molecular effects have been attributed to histone deacetylase inhibitors (HDACI's), including the induction of major histocompatibility (MHC) genes. Here we report that one FDA approved HDACI, Vorinostat, and a second HDACI currently in clinical trials, Entinostat, reduce the ratio of class II associated invariant peptide (CLIP) to the MHC class II molecule, HLA-DR, indicating an increase in the non-CLIP peptides bound to HLA-DR. The HDACI effects are apparent with immortalized B-cells, HLA-DR constitutive melanoma cells and with melanoma cells expressing HLA-DR due to transformation with an expression vector for the HLA-DR gene co-activator, CIITA. Entinostat treatment leads to upregulation of Cathepsin L1, and the HLA-DR peptidome of the Entinostat treated cells is consistent with increased Cathepsin L1 mediated proteolysis. These results indicate that HDACI treatments may alter the HLA-DR peptidome of cells in patients and provide a way to identify novel immunogens for vaccinations and the study of autoantigens.

Peak sweeping and gating using thermal gradient gas chromatography.

When axial temperature gradients are applied in gas chromatography (GC), i.e., "thermal gradient GC" (TGGC), the temperature changes both in time and position, T(t,L), along the column, allowing unique control of the movement and elution of sample components. One method of performing TGGC involves introducing a sample into a column with a preset decreasing temperature gradient along its length, waiting for a short time until the sample separates along the gradient, and then raising the temperature to sweep all of the compounds out of the column and into the detector (i.e., "peak sweeping"). This method of operation is demonstrated here using a simple laboratory apparatus based on simultaneous resistive heating and convective cooling. An experimental comparison between isothermal GC (ITGC), temperature programmed GC (TPGC) and TGGC shows that TGGC is essentially equivalent in performance to TPGC operation when using the same column length (peak capacity production rate of 106, 381 and 469 min(-1), respectively); however, narrower peaks and higher signal-to-noise are achieved in TGGC. Furthermore, TGGC helps to minimize band broadening and peak tailing that arise from column adsorption and less than perfect sample injection. The low thermal mass of the TGGC system allows rapid column heating (4000°C/min) and cooling (3500°C/min) for selective separation (i.e., "peak gating") of compounds in a mixture without sacrificing the resolution of earlier or later eluting compounds.