Remco Swart

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.

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.

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.

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.

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.

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.

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.

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

BackgroundGenome‐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.ResultsHere, 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.ConclusionsOur 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.

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.

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.