Agilent Manuals

The Agilent 1100 Series micro fraction collector is a high-performance solution for processing complex samples in applications such as proteomics, metabolomics, natural product research, and library generation. This Application Note describes the field of application for the Agilent 1100 Series micro fraction collector and discusses the features and software control of this device. In addition, the main modules of the micro fraction collection system are also discussed.

This application note describes the use of the Agilent RNA 6000 Pico assay for the assessment of genomic DNA contamination in total RNA isolated from embryonic kidney cells using laser capture microdissection (LCM). Integrity and relative concentration of total RNA were analyzed using the Agilent 2100 bioanalyzer and the RNA 6000 Pico assay. An unexpected peak between the 18S and 28S ribosomal RNA peaks was observed and unambiguously proven, by DNase digestion and genomic DNA spike-in experiments, to be caused by genomic DNA contamination. A simple on-column DNase digestion method is suggested to eliminate contaminating genomic DNA from total RNA samples obtained by LCM, fluorescent-activated cell sorting (FACS), or manual dissection.

This Application Note describes an improved method for protein identification with two-dimensional nano LC/MS, which utilizes the elution of digest peptides from a strong cation exchange column by an injected salt step gradient of increasing salt concentration with a subsequent reversed phase separation. However, in this approach the ion exchange chromatography does not produce its full performance. To improve the SCX chromatography performance a new method was developed, which works with a semi-continuous pumped salt gradient. This Application Note describes the improved method for two-dimensional nano LC/MS. To show the full performance of the method a complex tryptic digest of the yeast proteome was analyzed. The results obtained with the developed method are compared to the formerly used injection method as well as to an off-line 2D LC method.

A variety of heat-shock proteins (HSPs) were identified in E. coli grown under heat-shock conditions using Agilent Nanoflow Proteomics Solution.

This Application Note demonstrates the Agilent Nanoflow Proteomics Solution system1) as a powerful tool for analysis of proteomics samples. Using the approach of cation exchange, sample enrichment, reversed phase chromatography and nanospray ion trap mass spectrometry with subsequent database search, all of ten proteins were identified from a tryptic digestion model mixture and hundreds of proteins from a complex E. coli cell lysate.

This Application Note describes a method for increasing the resolution of complex proteome samples by using two-dimensional offline LC/MS. The method applies a continuous salt gradient in the first separation dimension, which can significantly increase the number of identified proteins from complex samples due to higher chromatographic resolution compared to stepwise elution. In addition, the method offers high flexibility for the investigator since micro fractions of the first dimension can be stored, re-analyzed or chemically modified prior to further analysis. To achieve optimal resolution, the applied 2D LC method strongly requires optimization of the two separation dimensions. Especially elution gradients, analysis time, stationary phase material, and column dimension need to be designed and assembled adequately to attain a considerable increase in overall peak capacity. This Application Note shows data which influences the variables and components. It can serve as a guideline for the investigator to adapt his separation method for a specific proteome sample in order to achieve maximum peptide sequence information by MS/MS analysis. This will result in a maximum of identified proteins after a database search.

Off-line multidimensional LC/MS can provide high resolution mass spectra, and improve protein identification

This application note describes a method to separate organic acids by ion suppression chromatography (ISC) using reversed-phase liquid chromatography columns and diode array detection. Experiments were performed using a new ZORBAX SB Aq column that successfully separated organic acids. Two standard mixtures of organic acids and a number of soft drinks and wines were analyzed.

This article discusses the application of mass spectrometry technology in protein research, focusing on the advantages and challenges of electrospray ionization combined with time-of-flight mass spectrometer for protein identification.

The paper introduces the application of LC/MS in drug monitoring, which can be used to analyze over 200 drugs, including identification and quantification.

Mass-based fraction collection of compound libraries using the Agilent 1100 Series purification system

This Application Note describes the optimization of the Agilent 1100 Series high throughput LC/MS system for different analytical tasks. It provides detailed instructions on how to optimize chromatography, diode-array and MSD detection to achieve highest sample throughput in combination with substance identification, how to acquire quantitative information while maintaining very high sample throughput, and how to achieve fast analysis of very low sample amounts.

This document describes a routine method for the quantitative determination of 4-nonylphenol (NP), 4-nonylphenol monoethoxylate (NP1EO), and 4-nonylphenol diethoxylate (NP2EO) in sludge samples. A Soxtec® extraction procedure was used to enrich the target compounds from the solid matrix. Quantitative determinations were performed by high-speed gas chromatography/mass spectrometry using a short apolar fused silica column. Derivatization allows NP1EO and NP2EO to be analyzed by gas chromatography. The relative standard deviation was close to 5% for the analysis of 10 different sludges analyzed seven times each. Recoveries were determined for a sludge reference material and were higher than 90%. The experimental limits of quantification were 2, 5, and 5 µg/g of dry matter (µg/g DM), respectively, for NP, NP1EO, and NP2EO. Good agreement was observed between results obtained with this method and those obtained by our previous method, which used normal-phase liquid chromatography with an aminosilica column. This method was applied to the determination of alkylphenols and alkylphenol mono- and diethoxylates in sewage sludge by high-speed gas chromatography/mass spectrometry.