The “front end” problem in Mass Spectrometry (MS) has long challenged researchers to devise sample processing strategies that convert complexity and contamination into robust analyte presentation. For modern proteomics, the staggering complexity and dynamic range of protein expression creates an immense front end and MS measurement challenge. In peptide analysis, multidimensional separations on-line1
now present thousands of peptides per hour to an MS instrument via Electrospray Ionization (ESI). The most common step before MS analysis by either ESI or MALDI is reversed-phase liquid chromatography (RPLC)2
which decontaminates and reduces sample complexity, with microcapillary LC/MS serving as an optimal way to introduce low-to-sub femtomole amounts3
of complex peptide mixtures.
As the field of MS-based proteomics continues to mature, many efforts are underway to increase both the number of proteins identified and the sequence coverage of each individual protein to enable better detection of mass discrepancies such as post-translational modifications (PTMs). Standard approaches typically enable 5–50% sequence coverage using MALDI-TOF–MS4
with greater coverage possible using either on-line nanobore LC–MS3
or off-line nanospray ESI–MS.5
Recently, Lubman and co-workers have reported that nearly 100% sequence coverage can be obtained by using MALDI-and-ESI-based methods together.6
Other recent strategies to the PTM measurement challenge involve targeted analysis of specific modifications (e.g., phosphorylation7–9
) or strategies to maximize the observation of small proteolysis products produced during enzymatic degradation.10,11
However, PTM detection and localization through 100% sequence coverage can be highly efficient when analyzing intact proteins directly by high-resolution tandem MS (MS/MS) in a top-down methodology.12
The processing of intact proteins presents a more difficult front end challenge for MS but also some advantages in the complete interrogation of DNA-predicted primary structure.
To realize a more general and robust implementation of the Top Down strategy, good control over undigested proteome samples becomes critical. Two-dimensional separations have been developed involving various chromatographic techniques, including ion exchange13–15
or size exclusion chromatography (SEC)16
with RPLC. Isoelectric focusing (IEF) with subsequent RPLC using nonporous silica (NPS) has been reported using electrospray ionization (ESI) and time-of-flight (TOF) MS for protein detection.17,18
in capillaries has been coupled to ESI–Fourier transform (FT) MS for protein profiling, but MS/MS for direct protein identification has only been accomplished with standard proteins and has not been achieved in a high-throughput setting to date.22
Typical amounts of total protein used range from 2 to 10 mg for solution phase 2-dimensional separations and mid-to-sub microgram for 1-dimensional, on-line capillary LC/MS.
Proteome-wide fractionation strategies for Top Down are labor intensive in both protein separations and MS analysis. A recent separation platform using preparative gel electrophoresis (PAGE) and RPLC coupled with an ESI/Q-FTMS instrument has made the “top down” approach more systematic via an offline processing platform.23
An acid labile surfactant (ALS)24,25
instead of SDS was utilized in the first dimension to provide strongly denaturing conditions for size-dependent proteome fractionation while avoiding tedious SDS removal prior to MS analysis. With an initial requirement of ~1 g of yeast cells, here we describe a smaller ALS-PAGE/RPLC platform that transfers samples more directly from RPLC to FTMS for more efficient sample-handling and MS/MS data acquisition. Such efforts to decrease sample utilization result in 15 or 300-fold improvements using a nanospray robot off-line or on-line capillary RPLC/FTMS, respectively, with improved sample and data processing augmenting an automated quadrupole-FT MS hybrid instrument.