Chromosome 11-centric human proteome analysis of human brain hippocampus tissue.

Human chromosome 11 is the third gene-rich chromosome having 1304 protein-coding genes. According to the GeneCards, this chromosome contains 240 genes related to diseases, as it is well known as a disease-rich chromosome. Although there are many protein-coding genes, the proteomic identification ratio is rather low. As a model study, human hippocampal tissues from patients suffering from Alzheimer's disease and epilepsy were prepared to evaluate the gene-centric statistics related to the gene expression and disorders of chromosome 11. A total of 8828 protein coding genes from brain tissues were extensively off-gel fractionated and profiled by a high resolution mass spectrometer with collision induced dissociation and electron transfer dissociation. Five-hundred twenty-three of the proteins from brain tissues were determined to belong to chromosome 11, representing 37% of the proteins reported in the Global Proteome Machine Database. We extracted gene clusters from a specific biological process or molecular function in gene ontology, among which the olfactory receptor genes showed the largest cluster on chromosome 11. Analysis of the proteome data set from the hippocampus provides a significant network associated with genes and proteins and leads to new insights into the biological and genetic mechanisms of chromosome 11-specific diseases such as Alzheimer's disease.

Shotgun lipidomics for candidate biomarkers of urinary phospholipids in prostate cancer.

Qualitative and quantitative profiling of six different categories of urinary phospholipids (PLs) from patients with prostate cancer was performed to develop an analytical method for the discovery of candidate biomarkers by shotgun lipidomics method. Using nanoflow liquid chromatography-electrospray ionization-tandem mass spectrometry, we identified the molecular structures of a total of 70 PL molecules (21 phosphatidylcholines (PCs), 11 phosphatidylethanolamines (PEs), 17 phosphatidylserines (PSs), 11 phosphatidylinositols (PIs), seven phosphatidic acids, and three phosphatidylglycerols) from urine samples of healthy controls and prostate cancer patients by data-dependent collision-induced dissociation. Identified molecules were quantitatively examined by comparing the MS peak areas. From statistical analyses, one PC, one PE, six PSs, and two PIs among the PL species showed significant differences between controls and cancer patients (p < 0.05, Student's t test), with concentration changes of more than threefold. Cluster analysis of both control and patient groups showed that 18:0/18:1-PS and 16:0/22:6-PS were 99% similar in upregulation and that the two PSs (18:1/18:0, 18:0/20:5) with two PIs (18:0/18:1 and 16:1/20:2) showed similar (>95%) downregulation. The total amount of each PL group was compared among prostate cancer patients according to the Gleason scale as larger or smaller than 6. It proposes that the current study can be utilized to sort out possible diagnostic biomarkers of prostate cancer.

Simultaneous profiling of lysophospholipids and phospholipids from human plasma by nanoflow liquid chromatography-tandem mass spectrometry.

In this study, an analytical method for the simultaneous separation and characterization of various molecular species of lysophospholipids (LPLs) and phospholipids (PLs) is introduced by employing nanoflow liquid chromatography-electrospray ionization tandem mass spectrometry (nLC-ESI-MS/MS). Since LPLs and PLs in human plasma are potential biomarkers for cancer, development of a sophisticated analytical method for the simultaneous profiling of these molecules is important. Standard species of LPLs and PLs were examined to establish a separation condition using a capillary LC column followed by MS scans and data-dependent collision-induced dissociation (CID) analysis for structural identification. With nLC-ESI-MS/MS, regioisomers of each category of LPLs were completely separated and identified with characteristic CID spectra. It was applied to the comprehensive profiling of LPLs and PLs from a human blood plasma sample and yielded identifications of 50 LPLs (each regioisomer pair of 6 lysophosphatidylcholines (LPCs), 7 lysophosphatidylethanolamines (LPEs), 9 lysophosphatidic acid (LPAs), 2 lysophosphatidylglycerols (LPGs), and 1 lysophosphatidylserine (LPS)) and 62 PLs (19 phosphatidylcholines (PCs), 11 phosphatidylethanolamines (PEs), 3 phosphatidylserines (PSs), 16 phosphatidylinositols (PIs), 8 phosphatidylglycerols (PGs), and 5 phosphatidic acids (PAs)).

Quantitative analysis of urinary phospholipids found in patients with breast cancer by nanoflow liquid chromatography-tandem mass spectrometry: II. Negative ion mode analysis of four phospholipid classes.

Analysis was performed on four different categories of phospholipids (phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG), and phosphatidic acid (PA)) from urine in patients with breast cancer. This quantitative analysis was conducted using nanoflow liquid chromatography-electrospray ionization-tandem mass spectrometry (nLC-ESI-MS-MS). This study shows the profiling of the phospholipids (PLs) that can be identified by the negative ion mode of MS. A previous study (Kim et al. Anal. Bioanal. Chem. 393:1649, 21) focused on only two PL classes: phosphatidylcholines (PCs) and phosphatidylethanolamines (PEs) and were identified by positive ion mode. PLs were extracted by lyophilization of 1 mL of urine from both healthy normal females and breast cancer patients before and after surgery. Separation of PLs was performed by nLC followed by structural identification of PLs using data-dependent collision-induced dissociation. A total of 34 urinary PL molecules (12 PSs, 12 PIs, four PGs, and six PAs) were quantitatively examined. Among the four PL categories examined in this study, most PL classes showed an increase in the total amounts in the cancer patients, yet PIs exhibited some decreases. The present study suggests that the lipid composition found in the urine of breast cancer patients can be utilized for the possible development of disease markers, when the analysis is performed with negative ion mode of nLC-ESI-MS-MS.

Quantitative analysis of phosphatidylcholines and phosphatidylethanolamines in urine of patients with breast cancer by nanoflow liquid chromatography/tandem mass spectrometry.

Phosphatidylcholines (PCs) and phosphatidylethanolamines (PEs) from urine of patients with breast cancer were qualitatively and quantitatively analyzed by nanoflow liquid chromatography-electrospray ionization tandem mass spectrometry (nLC-ESI-MS-MS). Urinary phospholipids (PLs) were extracted from three different categories of patients (non-cancer controls and breast cancer patients before and after surgery) by first lyophilizing only 1 mL of urine sample to enrich PLs. Next, nLC-ESI-MS-MS analysis of intact urinary phospholipids was performed, resulting in structural identification of 21 PCs and 12 PEs, followed by quantitative analysis using a multiple standard addition method. This study demonstrated that nLC-ESI-MS-MS can be powerfully utilized for the study of relative changes in the contents and concentration of urinary PCs and PEs from breast cancer patients: total concentration of PCs and PEs of patient sample increased to (144 +/- 9)% and (171 +/- 11)%, respectively, compared to control sample but they decreased significantly following surgery.

A simple and rapid method based on liquid chromatography-tandem mass spectrometry for the measurement of α-L-iduronidase activity in dried blood spots: an application to mucopolysaccharidosis I (Hurler) screening.

BACKGROUND: We developed an analytical method to measure α-L-iduronidase (IDUA) activity in dried blood spots. This was achieved by using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) with electrospray ionization in the positive ion mode. METHODS: Chromatographic separation was completed using mobile phase involving water-formic acid and acetonitrile-formic acid over 2.8 min of run time on a column with a Kinetex XB-C18 (Phenomenex, USA). The detection of column effluent was performed using a Xevo TQ-S triple quadrupole mass spectrometer (Waters, USA) in the multiple-reaction monitoring mode. This method was verified with blank and control samples at four activity levels: base, low, medium, and high. Control materials were provided from Centers for Disease Control and Prevention (CDC). RESULTS: Intra- and inter-day precisions were between 2.6% and 16.5% and between 7.9% and 17.0%, respectively. A correlative regression study on the IDUA activity in CDC-control samples performed to assess the validity of the developed method showed a highly significant linear association (r(2)=0.9976) between the calculated and CDC-reported values and an obvious difference in activity among the four levels. This reliable analytical method was applied to mucopolysaccharidosis I (Hurler) screening of patients under treatment (n=4) and in normal controls (n=129). IDUA activity ranged from 8.98 to 77.12 µmol/hr/L) in normal controls, and patients undergoing medical treatment showed low IDUA activity. CONCLUSIONS: This method had advantages of simplicity, rapid sample preparation, and liquid chromatographic separation, which efficiently inhibited ionization suppression induced by matrix effects in mass spectrometric detection.

Profiling of phospholipids in lipoproteins by multiplexed hollow fiber flow field-flow fractionation and nanoflow liquid chromatography-tandem mass spectrometry.

This study describes a coupled analytical method to carry out the systematic profiling of phospholipids (PLs) in high-density lipoproteins (HDL) and low-density lipoproteins (LDL) from human blood plasma. HDL and LDL of healthy human plasma samples were separated by size and collected on a semi-preparative scale using multiplexed hollow fiber flow field-flow fractionation (MxHF5). Phospholipid mixtures contained in the resulting HDL and LDL fractions were analyzed by shotgun nanoflow liquid chromatography-tandem mass spectrometry (nLC-ESI-MS-MS). We utilized a dual scan method for the separation and simultaneous characterization of complicated PL mixtures by nLC-ESI-MS-MS, such that phosphatidylcholine (PC) and phosphatidylethanolamine (PE) molecules were detected in positive ion mode in a first LC run. In a second LC run, phosphatidylinositol (PI), phosphatidylglycerol (PG), and phosphatidic acid (PA) were detected in negative ion mode. In this study, a total of 56 PLs from HDL and 52 PLs from LDL particles were characterized by their molecular structures from data dependent collision-induced dissociation (CID) experiments, and their relative abundances were compared.