Nano-flow High-performance Liquid Chromatography Research Articles

Application of porous layer open tubular capillary columns with narrow inner diameter in nanoflow-high performance liquid chromatography for N-derivatized amino acid enantiomers

Porous layer open tubular capillary columns with a narrow inner diameter (NPLOT) have potential in life science, especially in single-cell analysis. In this work, quinidine-based chiral NPLOT columns were prepared by in-situ thermal-initiated polymerization. The porous layer of the organic polymer was prepared with a 6 μm i. d capillary by in-situ thermal-initiated polymerization, following which the thickness and morphology of the NPLOT column were observed for different durations (3 h, 6 h, and 9 h) of polymerization. The thicknesses of the porous layer were 103±51 nm and 210±51 nm when polymerization was conducted for 3 h and 6 h, respectively. Subsequently, the columns prepared under 3 h of polymerization were applied in nanoflow-high performance liquid chromatography for the enantioseparation of N-derivatized amino acid enantiomers. Baseline separation was achieved in 2 min. The sample volume consumed was of the picoliter level, which would provide a means of research for single-cell analysis.

Proteomic Characterization of the Neural Ectoderm Fated Cell Clones in the Xenopus laevis Embryo by High-Resolution Mass Spectrometry.

The molecular program by which embryonic ectoderm is induced to form neural tissue is essential to understanding normal and impaired development of the central nervous system. Xenopus has been a powerful vertebrate model in which to elucidate this process. However, abundant vitellogenin (yolk) proteins in cells of the early Xenopus embryo interfere with protein detection by high-resolution mass spectrometry (HRMS), the technology of choice for identifying these gene products. Here, we systematically evaluated strategies of bottom-up proteomics to enhance proteomic detection from the neural ectoderm (NE) of X. laevis using nanoflow high-performance liquid chromatography (nanoLC) HRMS. From whole embryos, high-pH fractionation prior to nanoLC-HRMS yielded 1319 protein groups vs 762 proteins without fractionation (control). Compared to 702 proteins from dorsal halves of embryos (control), 1881 proteins were identified after yolk platelets were depleted via sucrose-gradient centrifugation. We combined these approaches to characterize protein expression in the NE of the early embryo. To guide microdissection of the NE tissues from the gastrula (stage 10), their precursor (midline dorsal-animal, or D111) cells were fate-mapped from the 32-cell embryo using a fluorescent lineage tracer. HRMS of the cell clones identified 2363 proteins, including 147 phosphoproteins (without phosphoprotein enrichment), transcription factors, and members from pathways of cellular signaling. In reference to transcriptomic maps of the developing X. laevis, 76 proteins involved in signaling pathways were gene matched to transcripts with known enrichment in the neural plate. Besides a protocol, this work provides qualitative proteomic data on the early developing NE.

Tryptic Peptides Bearing C-Terminal Dimethyllysine Need to Be Considered during the Analysis of Lysine Dimethylation in Proteomic Study.

Lysine methylation plays important roles in structural and functional regulation of chromatin. Although trypsin is the most widely used protease in mass spectrometry-based proteomic analysis for lysine methylation substrates, the proteolytic activity of trypsin on dimethylated lysine residues remains an arguable issue. In this study, we tested the ability of trypsin to cleave dimethylated lysine residues in synthetic peptides, purified albumin, and whole cell lysate, and found that the C-terminal of dimethylated lysine residue could be cleaved in a protein sequence-dependent manner. Kinetic studies revealed that the optimal digestion time and enzyme-to-substrate ratio for the cleavage of dimethylated lysine by trypsin was around 16 h and 1:50, respectively. We further showed the tryptic C-terminal lysine-dimethylated (C-Kme2) peptides could contribute to a significant portion of substrate identification in the proteomic study, which utilizes the chemical dimethylation labeling approach. More than 120 tryptic C-Kme2 peptides (7% of total peptides identified) were identified in chemically lysine-dimethyl-labeled HeLa whole cell lysate by a single-shot nanoflow high performance liquid chromatography with tandem mass spectrometry (nano-HPLC-MS/MS) analysis. Moreover, in an assay for substrate identification of protease Glu-C using stable isotope dimethyl labeling approach, our data showed the tryptic C-Kme2 peptides accounted for more than 13% of total tryptic peptides. Additionally, our in vivo methylome profiling data revealed some C-Kme2 peptides, which is of importance to identification and quantification of biologically relevant protein and lysine-methylated site. Therefore, we reason that the tryptic peptides bearing C-terminal dimethylated lysine need to be considered in the mass spectrometric analysis of lysine dimethylation.

Abstract 2203: A SRM/MRM based targeted proteomics strategy for absolute quantification of potential biomarkers of TKI sensitivity in EGFR mutated lung adenocarcinoma

Abstract Lung cancer remains the leading cause of cancer-related deaths worldwide. Epidermal growth factor receptor(EGFR) is the primary driver oncogene in 10-30% lung adenocarcinoma. Kinase domain mutations in EGFR (L858R and E746-A750 deletion) provide sensitivity to EGFR-specific tyrosine kinase inhibitors (TKIs). Despite good tumor inhibitory effects, after approximately a year, patients acquire resistance to EGFR TKIs. We have carried out a stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative mass spectrometry (MS) on a Thermo Orbitrap Elite to identify and characterize the dynamics of tyrosine phosphorylation upon TKI treatment of human lung adenocarcinoma cell lines. The degree of inhibition of phosphorylation of several phospho-sites correlated with the extent of TKI-sensitivity. Several kinases and adaptor proteins that were significantly differentially phosphorylated include AHNAK-Y160, -Y715, STAT5A-Y694, DAPP1-Y139, ERRFI1-Y394, NEDD9-Y164, NF1-Y2579, INNPL1-Y1135, suggesting that these are a subset of potential biomarkers of TKI response. To corroborate our results, the next logical step is to carry out clinical validation of these phospho-sites in human lung adenocarcinoma patients, however no absolute quantification assay is currently available for these tyrosine phosphorylated peptides in human samples. The MS method most often used to complement shotgun methods is selected/multiple reaction monitoring (SRM/MRM) because of its high sensitivity, speed and dynamic range. We have performed SRM/MRM-MS using a nano-flow high performance liquid chromatography instrument coupled to a nano-electrospray ionization source attached to a Triple Quadrupole MS (Agilent 6495). In this method, digested proteins are chromatographically separated and ionized. The resulting precursor ions are m/z filtered in the first quadrupole(Q1) and collision induced dissociation is carried out in Q2 and the resultant product ions are m/z filtered in Q3. Each precursor-product pair is termed as a transition. For absolute quantification, heavy isotope labelled versions of the target peptides are spiked in biological samples to build a calibration curve. For the robust assay development, we have carried out the initial empirical refinement process using the heavy labelled versions of the target peptides, including selection of optimal transitions, normalizing retention times of peptides, running peptide stability assays and digestion time course experiments. We are currently validating these assays in human lung adenocarcinoma cell lines with known EGFR mutations with or without treatment with 1st and 3rd generation EGFR TKIs. The overall goal of these targeted studies is to develop absolute quantitation assays to precisely and accurately quantify these potential biomarkers in clinical samples. Citation Format: Shivangi Awasthi, Xu Zhang, Tapan Maity, Benjamin L. Oyler, David R. Goodlett, Udayan Guha. A SRM/MRM based targeted proteomics strategy for absolute quantification of potential biomarkers of TKI sensitivity in EGFR mutated lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2203. doi:10.1158/1538-7445.AM2017-2203

Automated Trapping Column Exchanger for High-Throughput Nanoflow Liquid Chromatography.

As compared to conventional high-performance liquid chromatography (HPLC) techniques, nanoflow HPLC exhibits improved sensitivity and limits of detection. However, nanoflow HPLC suffers from low throughput due to instrument failure (e.g., fitting fatigue and trapping column failure), limiting the utility of the technique for clinical and industrial applications. To increase the robustness of nanoflow HPLC, we have developed and tested a trapping column exchanging robot for autonomous interchange of trapping columns. This robot makes reproducible, automated connections between the active trapping column and the rest of the HPLC system. The intertrapping column retention time is shown to be sufficiently reproducible for scheduled selected reaction monitoring assays to be performed on different trapping columns without rescheduling the selection windows.

Identification of proteins from 4200-year-old skin and muscle tissue biopsies from ancient Egyptian mummies of the first intermediate period shows evidence of acute inflammation and severe immune response.

We performed proteomics analysis on four skin and one muscle tissue samples taken from three ancient Egyptian mummies of the first intermediate period, approximately 4200 years old. The mummies were first dated by radiocarbon dating of the accompany-\break ing textiles, and morphologically examined by scanning electron microscopy of additional skin samples. Proteins were extracted, separated on SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) gels, and in-gel digested with trypsin. The resulting peptides were analysed using nanoflow high-performance liquid chromatography-mass spectrometry. We identified a total of 230 unique proteins from the five samples, which consisted of 132 unique protein identifications. We found a large number of collagens, which was confirmed by our microscopy data, and is in agreement with previous studies showing that collagens are very long-lived. As expected, we also found a large number of keratins. We identified numerous proteins that provide evidence of activation of the innate immunity system in two of the mummies, one of which also contained proteins indicating severe tissue inflammation, possibly indicative of an infection that we can speculate may have been related to the cause of death.This article is part of the themed issue 'Quantitative mass spectrometry'.

Liquid MALDI MS Analysis of Complex Peptide and Proteome Samples.

Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) is well-known to be a powerful technique for the analysis of biological samples. By using glycerol-based liquid support matrices (LSMs) instead of conventional MALDI matrices the power of this technique can be extended further. In this study, we exploited LSMs for the identification of complex samples, that is, the Lactobacillus proteome and a bovine serum albumin (BSA) digest. Liquid and solid MALDI samples were manually and robotically prepared by coupling a nanoflow high-performance liquid chromatography (nanoHPLC) system to an automated MALDI sample spotting device. MS and MS/MS data were successfully acquired at the femtomole level using TOF/TOF as well as Q-TOF instrumentation and used for protein identification searching sequence databases. For the BSA digest analysis, liquid MALDI samples resulted in peptide mass fingerprints, which led to a higher confidence in protein identification compared with solid (crystalline) MALDI samples; however, postsource decay (PSD) MS/MS analysis of both the proteome of Lactobacillus plantarum WCFS1 cells and BSA digest showed that further optimization of the formation and detection of peptide fragment ions is still needed for liquid MALDI samples, as the MS/MS ion search score was lower than that for the solid MALDI samples, reflecting the poorer quality of the liquid MALDI-PSD spectra, which can be attributed to the differences in PSD parameters and their optimization that is currently achievable.

Monitoring gradient profile on-line in micro- and nano-high performance liquid chromatography using conductivity detection

In micro- or nano-flow high performance liquid chromatography (HPLC), flow-splitters and gradient elutions are commonly used for reverse phase HPLC separations. When a flow splitter was used at a high split-ratio (e.g., 1000:1 or higher), the actual gradient may deviate away from the programmed gradient. Sometimes, mobile phase concentrations can deviate by as much as 5%. In this work, we noticed that the conductivity (σ) of a gradient decreased with the increasing organic-solvent fraction (φ). Based on the relationship between σ and φ, a method was developed for monitoring gradient profile on-line to record any deviations in these HPLC systems. The conductivity could be measured by a traditional conductivity detector or a capacitively coupled contactless conductivity detector (C4D). The method was applied for assessing the performance of an electroosmotic pump (EOP) based nano-HPLC. We also observed that σ value of the gradient changed with system pressure; a=0.0175ΔP (R2=0.964), where a is the percentage of the conductivity increase and ΔP is the system pressure in bar. This effect was also investigated.

Phosphorylated membrane proteome analysis of human neuroblastoma SH-SY5Y cell line

A strategy with the combination of multiprotease digestion and the selective enrichment of phosphopeptides by silica hybrid monolith based immobilized Ti4+ affinity chromatography (Ti4+ -IMAC) was proposed, and applied in the global profiling of phosphorylated membrane proteome of neuroblastoma SH-SY5Y cells. The fraction of membrane proteins was extracted by ultra speed centrifuge, followed by washing with 1 mol/L sodium chloride and 0.1 mol/L sodium carbonate. For digestion, chymotrypsin and pepsin with broader specificity were used as complementary enzymes to trypsin. The phosphopeptides were then selectively enriched by monolithic Ti4+ -IMAC column, and analyzed by nanoflow high performance liquid chromatography and mass spectrometry. A total of 43 phosphoproteins were identified, among which 14 proteins were located on the membrane. All these results demonstrated that the proposed strategy might be promising to promote the in-depth study of neuroblastoma and discover the candidate biomarkers.

High-pressure open-channel on-chip electroosmotic pump for nanoflow high performance liquid chromatography.

Here, we construct an open-channel on-chip electroosmotic pump capable of generating pressures up to ∼170 bar and flow rates up to ∼500 nL/min, adequate for high performance liquid chromatographic (HPLC) separations. A great feature of this pump is that a number of its basic pump units can be connected in series to enhance its pumping power; the output pressure is directly proportional to the number of pump units connected. This additive nature is excellent and useful, and no other pumps can work in this fashion. We demonstrate the feasibility of using this pump to perform nanoflow HPLC separations; tryptic digests of bovine serum albumin (BSA), transferrin factor (TF), and human immunoglobulins (IgG) are utilized as exemplary samples. We also compare the performance of our electroosmotic (EO)-driven HPLC with Agilent 1200 HPLC; comparable efficiencies, resolutions, and peak capacities are obtained. Since the pump is based on electroosmosis, it has no moving parts. The common material and process also allow this pump to be integrated with other microfabricated functional components. Development of this high-pressure on-chip pump will have a profound impact on the advancement of lab-on-a-chip devices.

Advancing the analysis of less specific proteolytic digests via nLC–ESI-QoTOF-MS/MS

Although less specific proteases have been established in proteomics, resulting peptides are often disadvantaged in mass spectrometric analysis compared to tryptic peptides. This is due to the high number of theoretically possible peptides as well as their relatively inefficient ionization and fragmentation. One possibility to improve the performance of less specific digests is to employ amine reactive, basic labels such as tandem mass tags (TMT) for the N-terminal derivatization of peptides. This study uses elastase and thermolysin digestion of cytosolic extracts with and without the use of TMT labeling for analysis via nanoflow high-performance liquid chromatography (nLC) coupled to an electrospray ionization quadrupole orthogonal acceleration time-of-flight (ESI-QoTOF) mass spectrometer. Differences between tryptic and less specific digests and their behavior during chromatographic separation and tandem mass spectrometric (MS/MS) analysis are highlighted. Furthermore, this work shows how the tandem mass spectrometric analysis of less specific digests can be optimized with reference to peptide ionization and fragmentation. The effect of TMT labeling on the number and characteristics of identified peptides as well as peptide fragmentation behavior is examined. Taking all these factors into consideration, this work constitutes an optimized strategy for the analysis of less specific digests via nLC–ESI-QoTOF-MS/MS.

Chip‐based nanoflow high performance liquid chromatography coupled to mass spectrometry for profiling of soybean flavonoids

In this work a chip-based nano HPLC coupled MS (HPLC-chip/MS) method with a simple sample preparation procedure was developed for the flavonoid profiling of soybean. The analytical properties of the method including the linearity (R(2) , 0.992-0.995), reproducibility (RSD, 1.50-7.66%), intraday precision (RSD, 1.41-5.14%) and interday precision (RSD, 2.76-16.90%) were satisfactory. Compared with the conventional HPLC/MS method, a fast extraction and analysis procedure was applied and more flavonoids were detected in a single run. Additionally, 13 flavonoids in soybean seed were identified for the first time. The method was then applied to the profiling of six varieties of soybean sowed at the same place. A clear discrimination was observed among different cultivars, three isoflavones, accounting for nearly 80% of total flavonoid contents, were found increased in the spring soybeans compared with the summer cultivars.

Comparison of standard- and nano-flow liquid chromatography platforms for MRM-based quantitation of putative plasma biomarker proteins

The analytical performance of a standard-flow ultra-high-performance liquid chromatography (UHPLC) and a nano-flow high-performance liquid chromatography (HPLC) system, interfaced to the same state-of-the-art triple-quadrupole mass spectrometer, were compared for the multiple reaction monitoring (MRM)-mass spectrometry (MS)-based quantitation of a panel of 48 high-to-moderate-abundance cardiovascular disease-related plasma proteins. After optimization of the MRM transitions for sensitivity and testing for chemical interference, the optimum sensitivity, loading capacity, gradient, and retention-time reproducibilities were determined. We previously demonstrated the increased robustness of the standard-flow platform, but we expected that the standard-flow platform would have an overall lower sensitivity. This study was designed to determine if this decreased sensitivity could be compensated for by increased sample loading. Significantly fewer interferences with the MRM transitions were found for the standard-flow platform than for the nano-flow platform (2 out of 103 transitions compared with 42 out of 103 transitions, respectively), which demonstrates the importance of interference-testing when nano-flow systems are used. Using only interference-free transitions, 36 replicate LC/MRM-MS analyses resulted in equal signal reproducibilities between the two platforms (9.3 % coefficient of variation (CV) for 88 peptide targets), with superior retention-time precision for the standard-flow platform (0.13 vs. 6.1 % CV). Surprisingly, for 41 of the 81 proteotypic peptides in the final assay, the standard-flow platform was more sensitive while for 9 of 81 the nano-flow platform was more sensitive. For these 81 peptides, there was a good correlation between the two sets of results (R(2) = 0.98, slope = 0.97). Overall, the standard-flow platform had superior performance metrics for most peptides, and is a good choice if sufficient sample is available.

Proteomic analysis of acquired tamoxifen resistance in MCF-7 cells reveals expression signatures associated with enhanced migration

IntroductionAcquired tamoxifen resistance involves complex signaling events that are not yet fully understood. Successful therapeutic intervention to delay the onset of hormone resistance depends critically on mechanistic elucidation of viable molecular targets associated with hormone resistance. This study was undertaken to investigate the global proteomic alterations in a tamoxifen resistant MCF-7 breast cancer cell line obtained by long term treatment of the wild type MCF-7 cell line with 4-hydroxytamoxifen (4-OH Tam).MethodsWe cultured MCF-7 cells with 4-OH Tam over a period of 12 months to obtain the resistant cell line. A gel-free, quantitative proteomic method was used to identify and quantify the proteome of the resistant cell line. Nano-flow high-performance liquid chromatography coupled to high resolution Fourier transform mass spectrometry was used to analyze fractionated peptide mixtures that were isobarically labeled from the resistant and control cell lysates. Real time quantitative PCR and Western blots were used to verify selected proteomic changes. Lentiviral vector transduction was used to generate MCF-7 cells stably expressing S100P. Online pathway analysis was performed to assess proteomic signatures in tamoxifen resistance. Survival analysis was done to evaluate clinical relevance of altered proteomic expressions.ResultsQuantitative proteomic analysis revealed a wide breadth of signaling events during transition to acquired tamoxifen resistance. A total of 629 proteins were found significantly changed with 364 up-regulated and 265 down-regulated. Collectively, these changes demonstrated the suppressed state of estrogen receptor (ER) and ER-regulated genes, activated survival signaling and increased migratory capacity of the resistant cell line. The protein S100P was found to play a critical role in conferring tamoxifen resistance and enhanced cell motility.ConclusionsOur data demonstrate that the adaptive changes in the proteome of tamoxifen resistant breast cancer cells are characterized by down-regulated ER signaling, activation of alternative survival pathways, and enhanced cell motility through regulation of the actin cytoskeleton dynamics. Evidence also emerged that S100P mediates acquired tamoxifen resistance and migration capacity.

Gel-free shotgun proteomic analysis of human milk

The composition of milk has adapted during the evolution of the species to fulfill the specific nutritional needs of the offspring. Currently, it is widely recognized that milk benefits go beyond mere nutrition and serve as a source of a number of functional components to the newborn, particularly host defense effectors. However, the human milk proteome description is still incomplete, primarily because the detection of low-abundance proteins remains challenging. To overcome the limitations of the classical electrophoresis-based approach, previously separated milk fat globule membrane (MFGM) and whey protein fractions were analyzed by nanoflow-high performance liquid chromatography (HPLC)/Fourier Transform-Ion Cyclotron Resonance (FT-ICR) mass spectrometry (MS). This shotgun strategy showed an as yet unmatched potential to profile low-abundance proteins in human milk. Proteins associated with 301 different gene products were identified, some of which could be clustered into subsets of protein isoforms, thus providing one of the largest protein inventories of human milk. The identified proteins, which were derived from multiple metabolic pathways, are involved in different physiological functions, such as membrane trafficking, cell signaling, fat metabolism and transport, metabolite delivery, protein synthesis/proteolysis or folding, and immunity-related actions. Nevertheless, it appears clear from this study that the overall picture of the human milk proteome is still incomplete, although several protein signatures of milk evolution are emerging.

Proteomics of exhaled breath: methodological nuances and pitfalls

The analysis of exhaled breath condensate (EBC) can be an alternative to traditional endoscopic sampling of lower respiratory tract secretions. This is a simple non-invasive method of diagnosing respiratory diseases, in particular, respiratory inflammatory processes. Samples were collected with a special device-condenser (ECoScreen, VIASYS Healthcare, Germany), then treated with trypsin according to the proteomics protocol for standard protein mixtures and analyzed by nanoflow high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) with a 7-Tesla Finnigan LTQ-FT mass spectrometer (Thermo Electron, Germany). Mascot software (Matrixscience) was used for screening the database NCBInr for proteins corresponding to the peptide maps that were obtained. EBCs from 17 young healthy non-smoking donors were collected. Different methods for concentrating protein were compared in order to optimize EBC preparations for proteomic analysis. The procedure that was chosen allowed identification of proteins exhaled by healthy people. The major proteins in the condensates were cytoskeletal keratins. Another 12 proteins were identified in EBC from healthy non-smokers. Some keratins were found in the ambient air and may be considered exogenous components of exhaled air. Knowledge of the normal proteome of exhaled breath allows one to look for biomarkers of different disease states in EBC. Proteins in ambient air can be identified in the respiratory tract and should be excluded from the analysis of the proteome of EBC. The results obtained allowed us to choose the most effective procedure of sample preparation when working with samples containing very low protein concentrations.

Quantitative analysis of a novel glucosylated phospholipid by liquid chromatography–mass spectrometry

Building upon the demonstrated presence of a new glyceroglycolipid, phosphatidylglucoside (PtdGlc), in rat embryonic brain tissues, we have developed a method to identify minute amounts of PtdGlc in cultured cells by using nano-flow high-performance liquid chromatography and negative-ion-mode electrospray linear-ion trap time-of-flight mass spectrometry (LC-MS). A normal-phase silica gel-based column enabled us to separate PtdGlc from other lipid classes. PtdGlc was identified from its tandem mass spectrometry spectrum and from its retention time in the column. Using an internal standard collection and LC-MS, we obtained the linearity of PtdGlc at a range of 6.3–800 fmol per injection. We applied this method to analyze quantitative changes in PtdGlc in C6 glioma cells after cellular differentiation into GFAP-positive glial cells. PtdGlc in C6 glioma cells consisted exclusively of C18:0/C20:0 fatty acyl chains. Differentiation induced by the addition of anti-PtdGlc antibody plus cAMP in culture medium significantly increased the glycolipid content.

Comparison of ESI-MS interfaces for the analysis of UV-crosslinked peptide–nucleic acid complexes

In this report, the effectiveness of high performance liquid chromatography (HPLC) in conjunction with electrospray ionization mass spectrometry (ESI-MS) is examined as a tool for identifying the sites of crosslinking in a protein that has been photoreacted with a non-photolabeled oligonucleotide. ESI-MS and MALDI-MS analyses preceded by off-line microflow and nanoflow HPLC, on-line microflow HPLC/ESI, and on-line nanoflow HPLC/ESI interfaces were performed in order to determine their relative effectiveness in separating mixtures of nucleopeptides and identifying sites of crosslinking on the individual components. The characteristics of these four techniques as well as possibilities for improving the analysis of nucleopeptides by ESI-MS are compared and discussed.

The Structure of Apolipoprotein A-II in Discoidal High Density Lipoproteins

It is well accepted that high levels of high density lipoproteins (HDL) reduce the risk of atherosclerosis in humans. Apolipoprotein A-I (apoA-I) and apoA-II are the first and second most common protein constituents of HDL. Unlike apoA-I, detailed structural models for apoA-II in HDL are not available. Here, we present a structural model of apoA-II in reconstituted HDL (rHDL) based on two well established experimental approaches: chemical cross-linking/mass spectrometry (MS) and internal reflection infrared spectroscopy. Homogeneous apoA-II rHDL were reacted with a cross-linking agent to link proximal lysine residues. Upon tryptic digestion, cross-linked peptides were identified by electrospray mass spectrometry. 14 cross-links were identified and confirmed by tandem mass spectrometry (MS/MS). Infrared spectroscopy indicated a beltlike molecular arrangement for apoA-II in which the protein helices wrap around the lipid bilayer rHDL disc. The cross-links were then evaluated on three potential belt arrangements. The data clearly refute a parallel model but support two antiparallel models, especially a "double hairpin" form. These models form the basis for understanding apoA-II structure in more complex HDL particles.