Aqueous size-exclusion chromatographic separations of intact proteins under native conditions: Effect of pressure on selectivity and efficiency.

The selectivity and separation efficiency of aqueous size-exclusion chromatographic separations of intact proteins were assessed for different flow rates, using columns packed with 3 and 5 µm silica particles containing 150 and 290 Å stagnant pores. A mixture of intact proteins with molecular weights ranging between 17 000 and 670 000 Da was used to construct the calibration curves. Both the model fit and the predictive properties, using a leave-one-out strategy, of different polynomial models (up to fifth order) were evaluated for different flow rates. The best compromise between model fit and predictive properties was obtained using a third-order polynomial model. The accuracy of the predictive properties decreased with 10% with an eightfold increase in the flow rate. No changes in retention factors (hence selectivity) were observed in the flow-rate range applied. A strong correlation between molecular weight and plate height was observed. Exclusion of large-molecular-weight proteins led to a significant reduction in the stationary-phase mass-transfer contribution to the total plate-height value, and this effect was also independent of the flow rate applied. The kinetic-performance limits, in terms of plate number and time, and optimal column-length particle-size combinations were determined at the maximum recommended operating pressure of the size-exclusion chromatography columns (20 MPa). Finally, the possibilities of method speed-up using ultra-high-pressure size-exclusion chromatography in combination with columns packed with sub-2 µm particles are discussed.

High-speed isocratic and gradient liquid-chromatography separations at 1500bar.

The need to improve either sample throughput on separation efficiency has spurred the development of ultra-high-pressure LC instrumentation, allowing to operate up to column pressures of 1500bar. In the present study, the isocratic and gradient performance limits were assessed at UHPLC conditions applying columns packed with core-shell particles. First, the extra-column band broadening contributions were assessed and minimized. Using an optimized system configuration minimum reduced plate heights of 1.8 were recorded on 2.1×100 columns packed with 1.5µm core-shell particles. Increasing the pressure limit from 500 to 1500bar and at the same time reducing the particle size from 2.6 to 1.5µm has allowed the analysis time to be decreased by a factor of 1.5 in isocratic mode, while maintaining separation efficiency (N=54,000). The kinetic time-gain factor in isocratic mode was proportional to the ratio of the separation impedance of both columns multiplied with the pressure ratio applied. In addition, the effect of operating pressure on the time gain factor was assessed in gradient mode. Using optimized gradient steepness (tG/t0=12) and increasing the operating pressure from 500 to 1500bar a time gain factor of almost 13 was achieved for the separation of a mixture of waste-water pollutants without compromising peak capacity.

Development and performance evaluation of an ultralow flow nanoliquid chromatography-tandem mass spectrometry set-up.

LC-MS/MS is the most commonly used technique for the identification and characterization of proteins. The efficiency of the electrospray process is a critical factor in LC-MS/MS. Despite the benefits associated with very low flow rates for the ionization efficiency, most LC-MS/MS platforms are operated at relatively high flow rates. The purpose of this work was to develop a nano LC system operable at a flow rate of 20 nL/min, applicable for routine analysis in proteomics laboratories. Peptide separation was performed with an analytical column packed with 2 µm porous chromatographic beads, a length of 25 cm and an inner diameter (i.d.) of 25 µm. Practical usability, reproducibility, and overall performance of the system were evaluated with a tryptic peptide mixture generated from HeLa cells. Using 100 ng of sample, we identified on average 3721 protein groups based on 25,699 peptides. We demonstrate that the number of peptides identified with this system increases with decreasing flow rates. Probing the sensitivity of the set-up we analyzed only 10 ng of the sample, identifying an average number of 2042 protein groups based on 11 424 peptides. All MS data have been deposited in the ProteomeXchange with identifier PXD000396 (http://proteomecentral.proteomexchange.org/dataset/PXD000396).

Transcriptome and proteome quantification of a tumor model provides novel insights into post-transcriptional gene regulation.

BACKGROUND: Genome-wide transcriptome analyses have given systems-level insights into gene regulatory networks. Due to the limited depth of quantitative proteomics, however, our understanding of post-transcriptional gene regulation and its effects on protein-complex stoichiometry are lagging behind. RESULTS: Here, we employ deep sequencing and the isobaric tag for relative and absolute quantification (iTRAQ) technology to determine transcript and protein expression changes of a Drosophila brain tumor model at near genome-wide resolution. In total, we quantify more than 6,200 tissue-specific proteins, corresponding to about 70% of all transcribed protein-coding genes. Using our integrated data set, we demonstrate that post-transcriptional gene regulation varies considerably with biological function and is surprisingly high for genes regulating transcription. We combine our quantitative data with protein-protein interaction data and show that post-transcriptional mechanisms significantly enhance co-regulation of protein-complex subunits beyond transcriptional co-regulation. Interestingly, our results suggest that only about 11% of the annotated Drosophila protein complexes are co-regulated in the brain. Finally, we refine the composition of some of these core protein complexes by analyzing the co-regulation of potential subunits. CONCLUSIONS: Our comprehensive transcriptome and proteome data provide a valuable resource for quantitative biology and offer novel insights into understanding post-transcriptional gene regulation in a tumor model.

High-speed gradient separations of peptides and proteins using polymer-monolithic poly(styrene-co-divinylbenzene) capillary columns at ultra-high pressure.

For the first time, polymer monolithic capillary columns have been employed at ultra-high-pressure liquid chromatographic conditions (UHPLC) to investigate their potential for high-speed separations of peptides and intact proteins. In comparison to conventional flow rates and gradient conditions, a substantial decrease in analysis time (>factor 4) can be achieved when operating monolithic columns such as ultra-high-pressure conditions while scaling the gradient volume. The effects of flow rate and column length on the peak capacity and the gradient performance limits were assessed for the separation of peptide and protein mixtures applying the maximum system pressure (80MPa) and a fixed gradient steepness. The potential for ultra-fast gradient separations of large biomolecules was further demonstrated for very steep gradients (gradient times≪1min). A tryptic digest of cytochrome c was separated using a gradient time of only 1min. Finally, the run-to-run repeatability and column robustness were assessed at ultra-high pressure conditions (after>800 runs) with consecutive steep 1min separations of peptides, yielding RSD values below 0.12% in retention time.

High-resolution peptide separations using nano-LC at ultra-high pressure.

We report on the optimization of nano-LC gradient separations of proteomic samples that vary in complexity. The gradient performance limits were visualized by kinetic plots depicting the gradient time needed to achieve a certain peak capacity, while using the maximum system pressure of 80 MPa. The selection of the optimal particle size/column length combination and corresponding gradient steepness was based on scouting the performance of 75 µm id capillary columns packed with 2, 3, and 5 µm fully porous silica C18 particles. At optimal gradient conditions, peak capacities up to 500 can be obtained within a 120 min gradient using 2 µm particle-packed capillary columns. Separations of proteomic samples including a cytochrome c tryptic digest, a bovine serum albumin tryptic digest, a six protein mix digest, and an Escherichia coli digest are demonstrated while operating at the kinetic-performance limit, i.e. using 2-µm packed columns, adjusting the column length and scaling the gradient steepness according to sample complexity. Finally, good run-to-run retention time stability (RSD values below 0.18%) was demonstrated applying ultra-high pressure conditions.

Analysis of protein mixtures from whole-cell extracts by single-run nanoLC-MS/MS using ultralong gradients.

The majority of proteome-wide studies rely on the high separation power of two-dimensional liquid chromatography-tandem mass spectrometry (2D LC-MS/MS), often combined with protein prefractionation. Alternative approaches would be advantageous in order to reduce the analysis time and the amount of sample required. On the basis of the recent advances in chromatographic and mass spectrometric instrumentation, thousands of proteins can be identified in a single-run LC-MS/MS experiment using ultralong gradients. Consequently, the analysis of simple proteomes or clinical samples in adequate depth becomes possible by performing single-run LC-MS/MS experiments. Here we present a generally applicable protocol for protein analysis from unseparated whole-cell extracts and discuss its potential and limitations. Demonstrating the practical applicability of the method, we identified 2,761 proteins from a HeLa cell lysate, requiring around 10 h of nanoLC-MS/MS measurement time.

Morphology and efficiency of poly(styrene-co-divinylbenzene)-based monolithic capillary columns for the separation of small and large molecules.

The morphology of organic monolithic stationary phases based on poly(styrene-divinylbenzene) was modified by changing the ratio of monomers to microporogen in order to make them also suitable for small molecule separations. The morphology of the columns was characterized by high-resolution scanning electron micrography, showing larger primary globules and larger macropores, as well as no mesopores >20 nm in the monolithic skeleton. The permeability of the modified monoliths was approximately three times higher than that of columns which have been optimized for large molecule separations, enabling operation of a 30 cm long column at pressures below 250 bar. In the isocratic separation of dansylated amino acids, plate counts of 50000-107000 m(-1) were achievable, which are equivalent to efficiencies obtained with 3.1 µm porous particles. The separation performance for small molecules in gradient elution was investigated using mixtures of dansylated amino acids, ß-lactam antibiotics, and thyroid hormones. Finally, the modified monolithic capillary columns also proved to be highly efficient in the separation of biopolymers such as peptides and proteins, enabling peak width at half height of 3-8 s and peak capacities of 110-180 in 15-30 min gradient runs.

Quality control in LC-MS/MS.

In the last 15 years, MS-based protein characterization has expanded at a rapid rate. This success is built upon constantly improving instrumentation and a variety of ingenious methods applied to numerous biological questions. However, the reproducibility of mass spectrometric results is considered by many as insufficient. In part, inadequate quality control might be responsible for the lack of reproducibility. Quality control is rarely discussed in scientific publications. Here, we briefly present measures undertaken in our laboratory to foster a general discussion of the subject.

Ultra-high-pressure RPLC hyphenated to an LTQ-Orbitrap Velos reveals a linear relation between peak capacity and number of identified peptides.

Currently, unbiased protein identification is mostly performed by directly coupling reversed-phase liquid chromatography (RPLC) via electrospray ionization to a mass spectrometer. In contrast to the innovations in mass spectrometric instrumentation, cutting-edge technology in RPLC has generally not been well adopted. Here, we describe the effects of increased peak capacities on the number of identified proteins and peptides in complex mixtures utilizing collision-induced dissociation on an LTQ-Orbitrap Velos, providing a rationale for using advanced RPLC technology in LC-MS/MS. Using two different column lengths and gradient times between 1 and 10 h, we found a linear relation between the obtained peak capacities and the number of identified peptides. We identified on average 2516 proteins in the tryptic digest of 1 µg of HeLa lysate using an 8 h gradient on a 50 cm column packed with 2 µm C18 reversed-phase chromatographic material.

Altered Mascot search results by changing the m/z range of MS/MS spectra: analysis and potential applications.

The Mascot search algorithm is one of the most commonly used tools for protein identification. Tandem mass spectrometry data searched against a protein sequence database is utilized for identifying peptides and proteins, each reported with a score. Higher Mascot scores are associated with lower chances of random hits. The process of peak selection performed by the search engine prior to the search is a critical aspect of the process. Here, we show that Mascot divides the MS/MS spectrum into fixed m/z regions for peak selection, starting at the lowest m/z value of the peak list. Therefore, modifying the m/z range of the peak lists by insertion of a dummy peak with low m/z value changes the ensemble of peaks used for searching. As a consequence, Mascot peptide scores and search results are altered significantly and a different subset of the peptides present in the sample is identified after processing. We further show that the effect can be exploited and additional proteins and peptides can be identified by repeating the search with a combined set of differently processed files, even when applying identical false-positive rates.

High-efficiency nano- and micro-HPLC--high-resolution Orbitrap-MS platform for top-down proteomics.

In terms of resolution, mass accuracy, and sensitivity, the Orbitrap represents one of the most potent mass analyzers available today. We here elucidate the potential of interfacing Orbitrap-MS to ion-pair RP HPLC for intact protein analysis. Using gradients of ACN and monolithic columns of 1.0 and 0.10 mm id, peak capacities between 120 and 130 were achievable within 20-25 min separation time. Compared with silica-based stationary phases, protein recovery and carryover from monolithic columns were found clearly superior. Intact proteins were detectable in a mass range covering 5.7-150 kDa with LODs in the low femtomol range. Compared with UV detection, MS detection with a scanning speed of 1.6 s per spectrum on average led to a 26% increase in chromatographic peak widths, whereas chromatographic patterns were mostly preserved in extracted ion chromatograms at an acquisition rate of 0.5 s per spectrum. Isotopic resolution of multiply charged ions was demonstrated for proteins up to 42 kDa. A micro-HPLC-Orbitrap-MS setup employing a 1.0 mm id column was utilized to characterize a 150 kDa recombinant monoclonal antibody. The applicability of nano-HPLC-Orbitrap-MS to the analysis of highly complex protein mixtures is demonstrated for the 70% ethanol extractable subproteome of wheat grains.

Selection of column dimensions and gradient conditions to maximize the peak-production rate in comprehensive off-line two-dimensional liquid chromatography using monolithic columns.

The peak-production rate (peak capacity per unit time) in comprehensive off-line two-dimensional liquid chromatography (LC/x/LC) was optimized for the separation of peptides using poly(styrene-co-divinylbenzene) monolithic columns in the reversed-phase (RP) mode. A first-dimension ((1)D) separation was performed on a monolithic column operating at a pH of 8, followed by sequential analysis of all the (1)D fractions on a monolithic column operating at a pH of 2. To obtain the highest peak-production rate, effects of column length, gradient duration, and sampling time were examined. RP/x/RP was performed at undersampling conditions using a short 10 min (1)D gradient. The peak-production rate was highest using a 50 mm long (2)D column applying an 8-10 min (2)D gradient time and was almost a factor of two higher than when a 250 mm monolithic column was used. The best way to obtain a higher peak-production rate in off-line LC/x/LC proved to be an increase in the number of (1)D fractions collected. Increasing the (2)D gradient time was less effective. The potential of the optimized RP/x/RP method is demonstrated by analyzing proteomics samples of various complexities. Finally, the trade-off between peak capacity and analysis time is discussed in quantitative terms for both one-dimensional RP gradient-elution chromatography and the off-line two-dimensional (RP/x/RP) approach. At the conditions applied, the RP/x/RP approach provided a higher peak-production rate than the (1)D-LC approach when collecting three (1)D fractions, which corresponds to a total analysis time of 60 min.

High-efficiency liquid chromatography-mass spectrometry separations with 50 mm, 250 mm, and 1 m long polymer-based monolithic capillary columns for the characterization of complex proteolytic digests.

In this study, high-efficiency LC-MS/MS separations of complex proteolytic digests are demonstrated using 50 mm, 250 mm, and 1m long poly(styrene-co-divinylbenzene) monolithic capillary columns. The chromatographic performance of the 50 and 250 mm monoliths was compared at the same gradient steepness for gradient durations between 5 and 150 min. The maximum peak capacity of 400 obtained with a 50mm column, increased to 485 when using the 250 mm long column and scaling the gradient duration according column length. With a 5-fold increase in column length only a 20% increase in peak capacity was observed, which could be explained by the larger macropore size of the 250 mm long monolith. When taking into account the total analysis time, including the dwell time, gradient time and column equilibration time, the 50mm long monolith yielded better peptide separations than the 250 mm long monolithic column for gradient times below 80 min (n(c)=370). For more demanding separation the 250 mm long monolith provided the highest peak production rate and consequently higher sequence coverage. For the analysis of a proteolytic digest of Escherichia coli proteins a monolithic capillary column of 1m in length was used, yielding a peak capacity of 1038 when applying a 600 min gradient.

Parameters affecting the separation of intact proteins in gradient-elution reversed-phase chromatography using poly(styrene-co-divinylbenzene) monolithic capillary columns.

An experimental study was performed to investigate the effects of column parameters and gradient conditions on the separation of intact proteins using styrene-based monolithic columns. The effect of flow rate on peak width was investigated at constant gradient steepness by normalizing the gradient time for the column hold-up time. When operating the column at a temperature of 60 degrees C a small C-term effect was observed in a flow rate range of 1-4 microL/min. However, the C-term effect on peak width is not as strong as the decrease in peak width due to increasing flow rate. The peak capacity increased according to the square root of the column length. Decreasing the macropore size of the polymer monolith while maintaining the column length constant, resulted in an increase in peak capacity. A trade-off between peak capacity and total analysis time was made for 50, 100, and 250 mm long monolithic columns and a microparticulate column packed with 5 microm porous silica particles while operating at a flow rate of 2 microL/min. The peak capacity per unit time of the 50mm long monolithic column with small pore size was superior when the total analysis time is below 120 min, yielding a maximum peak capacity of 380. For more demanding separations the 250 mm long monolith provided the highest peak capacity in the shortest possible time frame.

Automatic column coupling system to operate chromatographic supports closer to their kinetic performance limit and to enhance method development.

A new hardware solution is proposed that allows one to automatically change the length of a chromatographic bed. The setup is based on the serial coupling of chromatographic columns using two rotor-stator valves (with N positions, N + 1 ports). Despite the use of a prototype setup requiring rather long connection tubing, only 9% loss in efficiency is observed for compounds with a retention factor above 4 compared to the efficiency expected on the basis of the individual column results. It has been demonstrated for a number of isocratic and gradient separations that the system allows one to realize considerable analysis time savings by adapting the total column length to the specific sample requirements and/or to the stage of method development wherein one is working. During method development, a separation on a short column length can first be used to rapidly gain insight into the composition of the sample, leaving fewer runs to be done on a column of maximal length (offering efficiencies that are inaccessible with individual column systems). The ease with which information can be obtained on columns of different lengths can furthermore be exploited for screening purposes to detect coeluting components in a stage wherein they still appear completely unresolved (i.e., have a resolution well below R(s) = 0.5).

Accurate molecular weight analysis of histones using FFE and RP-HPLC on monolithic capillary columns.

Due to their large diversity with respect to post-translational modifications (PTMs), the family of histones provides a major analytical challenge in current proteomics research. Their function has a large impact on the transcription of DNA, and as a result, on the expression of proteins. The variation in PTMs regulates transcription, and, as a result, many methods are being employed for the in-depth analysis of histones. In this paper, we present a separation strategy for histones based on free-flow electrophoresis (FFE) followed by an RP separation on capillary monolithic PS-DVB columns. The capillary columns are directly interfaced with an FT-ICR MS providing an online system for the detection and accurate molecular weight analysis of intact histones.

1 mm ID poly(styrene-co-divinylbenzene) monolithic columns for high-peak capacity one- and two-dimensional liquid chromatographic separations of intact proteins.

The LC performance of a 1x50 mm polymer monolithic column format was demonstrated with high-peak capacity one- (1D) and offline two dimensional (2D) LC separations of intact proteins. After optimizing the RP 1D-LC conditions, including column temperature, flow rate and gradient time, a peak capacity of 475 was achieved within a 2-h analysis. The suitability of the monolithic column was also demonstrated for fast 1 min protein separations yielding 1 s peak widths determined at half peak height. In addition, an offline 2D-LC method was developed using the micro-fraction collection capabilities of the autosampler allowing automatic fractionation of intact proteins after the weak-ion-exchange (WAX) separation, and re-injection of the fractions onto the second-dimension RP monolithic column. The best peak capacity-to-analysis time ratio was obtained when applying 10 min second-dimension RP gradients. At optimized conditions, the WAX/x/RPLC separation of intact Escherichia coli proteins was performed within 6 h yielding a maximum theoretical peak capacity of 4880.

Optimizing the peak capacity per unit time in one-dimensional and off-line two-dimensional liquid chromatography for the separation of complex peptide samples.

To obtain the best compromise between peak capacity and analysis time in one-dimensional and two-dimensional (2D) liquid chromatography (LC), column technology and operating conditions were optimized. The effects of gradient time, flow rate, column temperature, and column length were investigated in one-dimensional reversed-phase (RP) gradient nano-LC, with the aim of maximizing the peak per unit time for peptide separations. An off-line two-dimensional LC approach was developed using a micro-fractionation option of the autosampler, which allowed automatic fractionation of peptides after a first-dimension ion-exchange separation and re-injection of the fractions onto a second-dimension RP nano-LC column. Under the applied conditions, which included a preconcentration/desalting time of 5 min, and a column equilibration time of 12.5 min, the highest peak capacity per unit time in the 2D-LC mode was obtained when applying a short (10 min) first-dimension gradient and second-dimension RP gradients of 20 min duration. For separations requiring a maximum peak capacity of 375, one-dimensional LC was found to be superior to the off-line strong cation-exchange/x/RPLC approach in terms of analysis time. Although a peak capacity of 450 could be obtained in one-dimensional LC when applying 120-min gradients on 500-mm long columns packed with 3-mum particles, for separations requiring a peak capacity higher than 375 2D-LC experiments provide a higher peak capacity per unit time. Finally, the potential of off-line 2D-LC coupled to tandem mass spectrometry detection is demonstrated with the analysis of a tryptic digest of a mixture of nine proteins and an Escherichia coli digest.

Performance of five different electrospray ionisation sources in conjunction with rapid monolithic column liquid chromatography and fast MS/MS scanning.

The performances of five different ESI sources coupled to a polystyrene-divinylbenzene monolithic column were compared in a series of LC-ESI-MS/MS analyses of Escherichia coli outer membrane proteins. The sources selected for comparison included two different modifications of the standard electrospray source, a commercial low-flow sprayer, a stainless steel nanospray needle and a coated glass Picotip. Respective performances were judged on sensitivity and the number and reproducibility of significant protein identifications obtained through the analysis of multiple identical samples. Data quality varied between that of a ground silica capillary, with 160 total protein identifications, the lowest number of high quality peptide hits obtained (3012), and generally peaks of lower intensity; and a stainless steel nanospray needle, which resulted in increased precursor ion abundance, the highest-quality peptide fragmentation spectra (5414) and greatest number of total protein identifications (259) exhibiting the highest MASCOT scores (average increase in score of 27.5% per identified protein). The data presented show that, despite increased variability in comparative ion intensity, the stainless steel nanospray needle provides the highest overall sensitivity. However, the resulting data were less reproducible in terms of proteins identified in complex mixtures -- arguably due to an increased number of high intensity precursor ion candidates.