The latest advances in instrumentation, sample-preparation techniques, and innovative technologies and hybrid applications were among the topics covered at the recent ASMS annual meeting in Denver.
Scientists from the University of Washington (Seattle) and New Objective discussed their use of a nanoLC-MS system to separate and ionize peptides and fragment, and isolate the ions to perform shotgun proteomics. The aim was to evaluate different column and gradient lengths on peak capacity, peptide identification, and suppression.
Edward Hsieh, post doc in the department of genome sciences at the University of Washington, described the advantages of using splitless nanoflow chromatography for separating complex peptide mixtures and the rationale behind the selection of unique peptide identifications and peak capacity as the variables for comparing outcomes when varying column and gradient lengths.
Hsieh noted the large amount of variability in the literature regarding these two chromatographic parameters, and his group hypothesized that longer columns would perform better. The Picofrit columns from New Objective used in the study ranged in length from 10 to 60 cm. The researchers used a nanoAcquity HPLC system (Waters) and a Thermo LTQ-FT Ultra (Thermo Fisher Scientific).
Each sample comprised 1 µg of C. elegans lysate and bovine serum albumin. Each study run compared four column lengths, three gradient lengths for each column length, and three analytical replicates for each of those combinations.
The researchers found that peak capacity increased linearly with column length, but there was not a similar correlation with unique peptide identifications. They hypothesized that the MS scan speed might not have been fast enough to take advantage of the enhanced chromatograph separation.
While the number of unique peptide identifications increased as column and gradient length increased, this was true only up to a point. For example, the 60 cm column showed little to no improvement over the 40 cm column; the 10 cm column run showed little improvement when going from a 60 to 90 minute gradient length versus from 30 to 60 minutes; and the 30 minute gradient run showed no improvement beyond a 10 cm column length.
Kenneth Lewis, Ph.D., CEO of OpAns, also presented work done in collaboration with New Objective. He described methods for steroid analysis using nanospray MS as “a work in progress.” The main advantage is the ability to use small sample volumes, which is particularly beneficial when working with pediatric samples or for serial sampling from small animal models such as mice. This requires high sensitivity in the pg/mL range.
Dr. Lewis's group used the Agilent 1200 SL liquid chromatograph coupled to an Agilent 6410 triple quadrupole HotBox mass spectrometer run in electrospray ionization (ESI) mode. They added an online automatic splitter from Analytical Scientific Instruments, which determines the high and low flow rates and automatically adjusts the flow to maintain the desired rate.
Although nanospray steroid assays are not yet better than conventional assays, they are improving, Dr. Lewis concluded. He described the nanospray technology they employed as robust and easy to use, with good sensitivity and about a 10-minute injection-to-injection interval.
Reid Brennen, a scientist from Agilent Technologies said that microfluidic devices for LC-MS applications provide low dispersion and minimize sample loss and can be readily integrated with a nano-LC column with an ESI contact. The microfluidic chips he described are stackable, with each layer providing additional functionality. He cited several benefits of microfluidics for LC-MS, including the potential for increased complexity and integrated workflows, reduced sample and media volumes, higher throughput, and shorter run times.
One challenge in designing the chips was the need for higher pressures and stronger materials to support larger columns. In 2010, Agilent developed a multilayer metal technology that made it possible to bond together metal chips. The company uses electrochemical etching to create ports, an LC column and various types of channels, transfer channels, frits, and an enrichment column/sample loop, for example.
The system now in development comprises an Agilent 1200 LC system, a modified Agilent 4240 Chip Cube, an Agilent 6520 Q-TOF MS, and the experimental metal chip devices. In his talk, Brennen described the analysis of a BSA digest using this system and reported good or better sequence coverage as compared to a polyimide chip, without loss of peptides.