Improved Reversed Phase Chromatography of Hydrophilic Peptides

Technical Note pubs.acs.org/jpr

Improved Reversed Phase Chromatography of Hydrophilic Peptides from Spatial and Temporal Changes in Column Temperature Clifford Young,*,† Alexandre V. Podtelejnikov,‡ and Michael L. Nielsen*,† †

The Novo Nordisk Foundation Center for Protein Research, Proteomics Program, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, DK-2200 Copenhagen, Denmark ‡ Evosep Biosystems, Thriges Plads 6, DK-5000 Odense, Denmark S Supporting Information *

ABSTRACT: Reversed phase chromatography is an established method for peptide separation and frequently coupled to electrospray ionization−mass spectrometry for proteomic analysis. Column temperature is one parameter that influences peptide retention and elution, but it is often overlooked as its implementation requires additional equipment and method optimization. An apparatus that allows temperature manipulation in three areas of a twocolumn setup was evaluated for improvements in chromatography. Using commercially available standards, we demonstrate that a low column temperature (0 °C) during sample loading enhances the peak shape of several bovine serum albumin hydrophilic peptides. For digested HeLa lysates, approximately 15% more peptide identifications were obtained by increasing the precolumn temperature to 50 °C after the 500 ng sample was loaded at a low temperature. This method also identified additional early eluting peptides with grand average of hydropathicity values less than −2. We also investigated the effect of cooler column temperatures on peptides with post-translational modifications. It was possible to minimize the coelution of an isoaspartylated peptide and its unmodified version when the analytical column temperature was decreased to 5 °C. Aside from demonstrating the utility of lower temperatures for improved chromatography, its application at specific locations and time points is critical for peptide detection and separation. KEYWORDS: hydrophilic peptide, isoaspartic acid, liquid chromatography, mass spectrometry, temperature

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INTRODUCTION Bottom-up proteomic approaches typically involve mass spectrometric analysis of peptides produced from the tryptic digestion of proteins.1 Because sample digestion can produce a large number of peptides with similar physicochemical properties, reversed phase chromatography is often used to reduce undersampling by minimizing the simultaneous introduction of electrosprayed peptide ions into the mass spectrometer.2 Reversed phase chromatography involves introducing the sample in aqueous conditions onto a column containing a nonpolar stationary phase, so that when the organic solvent concentration is increased, analytes are eluted in order of increasing hydrophobicity. In addition to offline3 and online4 sample prefractionation, analytical columns with higher peak capacity5−7 can be employed to achieve deeper proteomic coverage of complex samples. Since every aspect of the liquid chromatography (LC) system contributes to the quality of the final peptide separation, the optimization of a small number of parameters can be difficult to perform in a comprehensive and timely manner. As the analytical column is arguably one of the most important factors in bottom-up proteomic experiments, it is not unusual that when improvements in chromatography are desired, column specifications such as length, particle size and internal diameter are preferentially investigated.8,9 Conversely, column temperature © XXXX American Chemical Society

is generally overlooked in liquid chromatography−mass spectrometry (LC−MS) due to the requirement of additional equipment and concern about elevated temperatures promoting siloxane bond hydrolysis, which decreases the stability of the stationary phase.10 Despite these issues, numerous chromatographical benefits can be obtained when the column temperature is increased to a stable level above room temperature, such as better peak selectivity and resolution.11 Not only ambient temperature fluctuations that interfere with the reproducibility of chromatography runs are eliminated, but improved mass transfer leads to better column efficiency, while shorter retention times allows the possibility of higher sample throughput.12 Moreover, the reduced viscosity of the mobile phase at higher temperatures leads to a reduction in backpressure, which opens up the possibility of further improvements that may not have been feasible under ambient conditions, such as using a longer column or smaller particles.13 It is therefore common practice that if the analytical column temperature can be altered, it is invariably increased. In contrast, investigations into subambient column temperatures for better chromatographical separations have been studied with small molecules14−16 and occasionally peptiReceived: December 20, 2016

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DOI: 10.1021/acs.jproteome.6b01055 J. Proteome Res. XXXX, XXX, XXX−XXX

Technical Note

Journal of Proteome Research

Figure 1. Overview of DTF setup. (A) Precolumn and analytical column temperatures can be modulated in spatial (dashed areas 1, 2 and 3) and temporal contexts (purple areas denote sample loading and elution, inset) to influence peptide chromatography. Some details (DTF covers and LC− MS components) have been omitted for clarity. (B) On the basis of the location of the three temperature controlled areas, a notation system consisting of three squares was developed to represent the methods. (C) Overview of the 15 column temperature methods used in this study. The color(s) within each square denotes the local temperature conditions. In some cases, specific sample loading and gradient conditions are described.

des.17−20 In either case, the analytical strategy involves the use of a lower temperature during sample loading, which encourages the trapping of analytes into a narrower band toward the proximal end of the column, hereby reducing the dispersion of analytes. When a superambient column temperature is performed to promote an efficient elution, an improvement in sensitivity should be observed for analytes concentrated by the chilled column temperature. Various apparatuses that can perform the thermal focusing of analytes have been developed. For example, an oven was constructed where the column could be physically moved into separate cold and hot zones to perform temperature-assisted loading and elution, respectively.14 Eghbali et al. demonstrated improved trapping of cytochrome c peptides due to chilling and subsequent heating by a cooling chip, although it should be noted that the temperature changes were performed near the distal end of the capillary column.17 A Peltier element

positioned at the column inlet was used to create the subambient and elevated temperature environments that resulted in better sensitivity of the neuropeptide galanin18 and early eluting bovine serum albumin (BSA) tryptic peptides.19 Further developments involved the use of multiple Peltier elements to perform successive cycles of temperatureassisted solute focusing to improve the chromatography of small molecules.16 The use of chilled buffer for the loading of samples while the precolumn and analytical column were heated led to higher BSA sequence coverage, as well as increases in the number of identified urinary peptides and proteins.20 It is important to mention that all these studies used fused silica capillary columns, which have low thermal mass and high thermal conductivity properties that promote rapid and efficient temperature manipulations, respectively.21 Although most proteomic LC−MS separations are performed with fused silica columns, the effects of thermal B


Technical Note


volume that exists between the columns. However, the orthogonal mounting of the DTF device and the requirement that the outlet of the analytical column must be positioned adjacent to the mass spectrometer orifice means that the distal half of the analytical column is unable to be subjected to temperature control. The collective aim of this study was to investigate the effect of different temperature environments on the reversed phase chromatography of various peptide mixtures. By adjusting column temperatures with respect to both location and time, benefits in chromatography were observed in samples differing in complexity and those possessing an isoaspartic acid posttranslational modification. Chilling of the precolumn during sample loading increased the peak intensity of several BSA hydrophilic peptides, while other hydrophilic peptides benefitted from the presence of the ion-pairing reagent trifluoroacetic acid (TFA). HeLa peptides loaded in the presence of a cooled precolumn (prior to elution with high temperature) produced an increase in the number of identified peptides, although another method that focused peptides on the precolumn and then at the beginning of the analytical column generated the highest number of early eluting hydrophilic peptides. Biases in the grand average of hydropathicity (GRAVY) scores of early eluting hydrophilic peptides were also observed between the two HeLa methods. Finally, analytical column chilling improved the resolution of two peptides differing in their isoaspartylation status, thus allowing both peaks to be quantified more accurately. These observations not only demonstrate that subambient temperatures contribute to chromatographic improvements, but that they also extend the capabilities of reversed phase chromatography. We envisage that numerous proteomic analyses can benefit from the temporal implementation of subambient column temperatures.

focusing on proteomic analyses have not been extensively studied. Because of their poor retention to the C18 column material, hydrophilic peptides are notoriously difficult to both observe and separate with reversed phase chromatography. They are also highly susceptible to band broadening caused by volume overload.18 We were not only interested in whether a subambient column temperature could transiently increase the retention of hydrophilic peptides during sample loading, but also if an elevated column temperature during elution would produce further improvements in their peak shape. After the construction of a “dynamic thermal focusing” (DTF) device that permits temperature control in three distinct locations (area 1, precolumn; area 2, inlet of the analytical column and area 3, middle of the analytical column) of a two-column setup (Figure 1A), experiments were conducted to determine the effect of different temperatures on the retention and elution of peptides from samples of varying complexity (Figures 1B and 1C). We also considered whether temperature controlled columns could provide any chromatographical benefit for peptides containing post-translational modifications, especially those that have a propensity to coelute with their unmodified counterparts. As an example, aspartic acids are susceptible to isomerization that results in an isobaric isoaspartic acid modification, where a methylene group from the side chain of the unmodified form is incorporated into the peptide backbone.22 This modification can affect the conformation and function of proteins,23,24 so the ability to detect and determine the modification site is important. However, these modified peptides can be difficult to analyze by LC−MS since coelution with its unmodified variant can occur with reversed phase chromatography, while the mass spectrometer is unable to discriminate between the two isoforms as there is no mass difference between them.25 Various strategies have been developed to identify isoaspartic acid containing peptides, which include the AspN digestion of samples to enrich for modified peptides26 and the use of electrostatic repulsionhydrophilic interaction chromatography25 for better peptide separation. Many LC−MS analyses inherently rely on tandem mass spectrometry (MS/MS) spectra from modified peptides fragmented by electron capture dissociation27,28 or electron transfer dissociation,29 which produce c+57 and z•−57 ions that reveal the modification site. Nonetheless, these analyses still require chromatographical separation of the unmodified and modified peptides, as coelution can generate MS/MS spectra with fragment ions produced from both species. Hence, it would be advantageous if one could chromatographically separate isoaspartylated peptides from their unmodified versions without the need for extensive sample preparation or major chromatographical changes. Compared to the devices that perform temperature manipulations on a one-column system,14−19 the combination of a two-column setup with several temperature controlled areas should provide several advantages. The demarcation of columns makes it easier to assess the impact of temperature changes on sample loading. Because samples are loaded onto a shorter precolumn, higher flow rates can be used during loading to reduce analysis time and increase sample throughput. This difference becomes more apparent when the viscosity of the mobile phase increases during column cooling. It shares design similarities with another device,16 where the compartmentalization of the DTF apparatus allows the cold trapping of analytes for a second time at the proximal end of the analytical column, where cooler temperatures can also minimize the effect of dead

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EXPERIMENTAL SECTION

Peptide Standards

Tryptic digests of BSA was obtained from Bruker Daltonik GmbH. HeLa protein digest standards and 12 synthetic peptides for isoaspartic acid chromatographical analysis (Table S1, purity >97%) were purchased from Thermo Scientific. The selection of synthetic peptides was based on LC−MS studies reporting the elution of identified aspartic and isoaspartic acid containing peptides as either concurrent or with similar retention times.25,28 LC Setup for DTF Analyses

All reversed phase chromatography was conducted on an EASY-nLC II system (Thermo Scientific, Odense, Denmark) with solvent A composed of 0.5% acetic acid and solvent B containing 80% acetonitrile in 0.5% acetic acid. The online twocolumn system consisted of a 2 cm precolumn (100 um inner diameter packed with 5 um C18 particles) obtained from Thermo Scientific and a 15 cm analytical column (75 um inner diameter packed in-house with 3 um C18 particles). ReproSilPur 120 C18-AQ particles from Dr. Maisch (Ammerbuch, Germany) were utilized in both columns. Briefly, the DTF apparatus is composed of three aluminum blocks that has grooves to accommodate the 360 um outer diameter capillaries and accompanying fittings. The columns are held in place by aluminum covers that can be fastened with thumbscrews, which also permits the tip of the analytical column to be positioned in front of the mass spectrometer orifice. Peltier elements that C


Technical Note


exclusion was increased to 30 s. All temperature methods were conducted in duplicate.

provide localized temperature control via heating (approximately 0.67 °C/s) or cooling (approximately −0.17 °C/s) are sandwiched between the aluminum blocks and stainless steel heatsinks. To minimize energy loss to the outside environment, layers of insulating foam are used to surround the aluminum blocks. The apparatus allows a large range of temperatures to be set (−10 to 70 °C) during the chromatographical method, thus providing the ability for any temperature controlled area to be subjected to a constant temperature (Figures S1A and S1B) or multiple temperature changes (Figure S1C). Additional information can be obtained from United States patent 8613216. The subambient and elevated temperatures used in this study were not as extreme as reported in other studies,18,19 but this was implemented to minimize any possible effects of temperature-induced stress on the C18 particles.

LC−MS Analysis of Isoaspartylated Peptides

Peptide stock solutions were prepared by adding water and performing sonication for 10 min, with aliquots (1 mg/mL) stored at −20 °C. Prior to LC−MS analysis, single peptides or peptide mixtures were diluted in solvent A to load approximately 208 pg of each peptide onto the precolumn. Chromatography was performed under similar conditions as the earlier BSA runs, except that the gradient length was extended to 20 min. Because of the higher backpressure encountered with methods involving cooling the middle of the analytical column, the re-equilibration flow rate was reduced to 400 nL/min to ensure these LC−MS runs could be performed without interruption. Mass spectra were acquired in a similar manner as the BSA samples, but dynamic exclusion was set to zero due to the possible coelution of an aspartic acid containing peptide with its corresponding isoaspartic acid isomer. The chromatographic analysis was conducted with triplicate runs. Retention time and peak area calculations were performed with Xcalibur software version 3.0.63 (Thermo Scientific) using the Genesis peak detection algorithm.

LC−MS Analysis of BSA Peptides

Tryptically digested BSA (10 fmol/uL) was prepared in solvent A. Prior to sample loading, the three temperature controlled areas were set to 25 °C and the columns equilibrated at 250 bar with 5 uL of solvent A. In conjunction with the temperature settings specified in each method, samples (5 uL) were loaded onto the precolumn at 250 bar (unless stated otherwise) in solvent A. For TFA experiments, BSA samples were instead loaded in the presence of 0.1% TFA (Figure S1D). BSA gradients (0 to 40% solvent B) were performed at a flow rate of 250 nL/min for 10 min, which was later increased to 100% solvent B over 5 min. Columns were washed in solvent B for 8 min while the flow rate was doubled. Re-equilibration of the columns started with a 2 min transition to solvent A before a final 5 min step in pure solvent A. It should be noted that the length of time from sample loading to gradient start can vary between the temperature methods due to the effect of temperature on solvent viscosity and the requirement that the desired temperature must be reached before the programmed chromatographical event can begin. Mass spectra was acquired on a Q Exactive Plus (Thermo Scientific, Bremen, Germany) configured with a 2 kV spray voltage and ion transfer capillary heated to 275 °C. Full scans (m/z 300 to 1750) were obtained in positive ion mode at 70000 resolution (m/z 200) with an automatic gain control (AGC) target of 1 × 106. If the intensity of multiply charged peptides surpassed the intensity threshold of 1 × 105, up to a maximum of five different precursors were individually isolated within a 1.3 m/z window for fragmentation by higher energy collisional dissociation (normalized collision energy of 25). MS/MS scans were acquired at 35000 resolution using an 1 × 106 AGC target (120 ms maximum injection time). All spectra were acquired in profile mode and dynamic exclusion was set to two seconds. Triplicate runs were performed for each BSA temperature method.

Peptide and Protein Identification

BSA raw files were processed with MaxQuant version 1.5.1.2, with database searches conducted against the BSA Uniprot entry (P02769, October 2014). The enzyme specificity was set to Trypsin/P and a maximum of two miscleavages were permitted. The minimum peptide length was lowered to five residues. N-terminal protein acetylation and methionine oxidation were specified as variable modifications, while cysteine carbamidomethylation was included as a fixed modification. Peptide tolerances for the first and main searches were 20 and 4.5 ppm, respectively. The MS/MS tolerance was set to 20 ppm, while other relevant settings included disabling of the second peptides option and selection of the calculate peak properties option. The scoring of peptides was performed by the integrated Andromeda search engine.30 MaxQuant searches of HeLa runs were performed against a human Uniprot database (88812 protein sequences, October 2014) that also contained reverse sequences and protein sequences from frequently found contaminants. The majority of search settings from the BSA analysis were also employed for the HeLa runs, except that a 1% false discovery rate was used to limit the number of peptide and protein identifications, while a minimum of two peptides was required for protein identification.

LC−MS Analysis of HeLa Peptides

Retention Time and Retention Length Comparisons of Hydrophilic HeLa Peptides

HeLa samples for LC−MS analysis were diluted to 100 ng/uL in solvent A. Sample loading and peptide chromatography were performed with the same settings as specified for the BSA samples, except that the gradient was conducted over 2 h. Mass spectra were also acquired on a similarly configured Q Exactive Plus, but changes were implemented to address the higher sample complexity, such as the AGC target increasing to 3 × 106 for full scans. Up to 12 different peptide precursors could be isolated for fragmentation (2 m/z window) within a duty cycle and MS/MS scans were acquired at 17500 resolution (40 ms maximum injection time). Accordingly, the dynamic

Retention times and retention lengths (peak width at base) were compiled for unmodified HeLa peptides possessing the highest scoring peptide spectrum match in each LC−MS run. For an unmodified peptide to be considered representative of a temperature method, it was required that it had to be identified in both runs. The average retention times of these peptides were then binned into five groups, which was based on the average retention times of four contaminating BSA peptides identified from the same LC−MS runs (Table S2). Peptides detected within the earliest time period were regarded as early eluting peptides. D


Technical Note


Figure 2. Effect of precolumn cooling and ion-pairing on BSA peptides CCTKPESER, ATEEQLK and SEIAHR. (A−C) Overlaid extracted ion chromatograms of the respective BSA peptides obtained with control (black), precolumn cooled (blue) and 0.1% TFA loaded control (green) temperature methods. (D−F) Peak area quantification of the aforementioned chromatograms. Each temperature method was performed in triplicate (closed circles), with hollow bars representing the sample means.

GRAVY Score Calculation

S2). These BSA peptides, which included one of the earliest eluting peptides appearing in the controls (TCVADESHAGCEK, where C is a carbamidomethylated cysteine), were largely unaffected by changes in column temperature, with only small differences observed in peak area (Figures S3A to S3E), retention time (Figures S4A to S4E) and peak width (Figures S5A to S5E). The vast majority of BSA peptides also exhibited a similar pattern of observations where only minor differences were detected. The performance of the DTF apparatus was evaluated by examining whether the retention times of the selected BSA peptides were affected by the temperature methods in a predictable manner. As expected, lower constant temperatures increased the retention times compared to the control method (Figures S4A to S4E), with cooling of the largest analytical column area producing the most severe change. Heat exposure to the precolumn generally resulted in the earlier elution of peptides, but constant analytical column heating methods did not reduce the elution times of the representative peptides. This suggests that additional heating coverage of the analytical column may be required to produce the beneficial chromatographical effects commonly associated with elevated temperatures. In the case where BSA peptides were loaded onto the precolumn maintained at a subambient temperature (0 °C), several high intensity peaks appeared early in the gradient. One peak was identified as CCTKPESER and classified as a moderately hydrophilic peptide due to its GRAVY score of −1.50. The extracted ion chromatogram from a control run demonstrates the poor retention of CCTKPESER to the stationary phase, with low signal appearing from near the analytical column dead time until its final elution (Figure 2A). Conversely, at a slightly later time point, the same peptide

GRAVY peptide scores were calculated by summing the hydropathy values of each amino acid in the sequence before division by the number of residues present.31 Hydrophilic peptides were defined as peptides possessing GRAVY scores less than zero, with classification of their hydrophilicity strength based upon the score (< 0 to −1 as weakly hydrophilic, < −1 to −2 as moderately hydrophilic and < −2 as strongly hydrophilic). Discrimination Factor Calculation

Discrimination factors that quantify the separation between adjacent peaks were calculated as previously described.32 A discrimination factor of 0 indicates the absence of a valley, while a discrimination factor of 1 indicates the signal is at the baseline between peaks. Data Presentation

Univariate scatterplots were produced instead of bar graphs to disclose raw data,33 while area-proportional Venn diagrams were created with the assistance of eulerAPE version 3.34

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RESULTS AND DISCUSSION

Effect of Column Temperatures on BSA Peptides

To determine the influence of column temperature on peptide chromatography, tryptically digested BSA peptides were separated by reversed phase chromatography on the DTF setup. Both columns were maintained at room temperature (25 °C) in the control method, while the other methods specified temperature changes that could be performed at three distinct areas (Figure 1C). On the basis of the range of observed retention times from control runs, we selected five BSA peptides to closely monitor throughout the experiment (Figure E


Technical Note


LC−MS Comparison of Column Temperatures against Ion-Pairing

eluted as a well resolved and intense peak under chilled precolumn conditions. The increase in peak area compared to the control runs was approximately sixty-fold (Figure 2D). Another peptide that benefitted from the chilled temperature conditions was identified as ATEEQLK (GRAVY score −1.36). Although this peptide eluted as a small resolved peak in a control run (Figure 2B), a 10-fold increase in peak area was regularly obtained with the cooled precolumn (Figure 2E). These examples demonstrate the advantage of using lower temperatures for the improved retention of hydrophilic peptides. No large increases in peak area were obtained for CCTKPESER (Figure S6A) and ATEEQLK (Figure S6B) with precolumn heating or when other constant temperature methods were exclusively performed on the analytical column. Further optimization of peptide chromatography was attempted with a dynamic temperature method, which builds upon the advantages of precolumn cooling but with two important methodological alterations. Not only was the cooling of the precolumn restricted to the duration of sample loading, but the precolumn was heated to 50 °C at the start of the gradient to minimize analyte broadening and encourage efficient peptide elution. Intriguingly, the increase in peak area of both aforementioned peptides was halved in these conditions compared to precolumn cooling alone (Figures S6A and S6B), with the CCTKPESER peptide eluting extremely early as a very broad peak with low intensity (Figure S6C). We hypothesized that its peak shape could be improved by prolonging the lower temperature conditions prior to precolumn heating. Since all methods specify the same volume to be loaded onto the precolumn, any decrease in the 250 bar maximum backpressure limit requires the loading duration to be extended as compensation. Although the peak width of CCTKPESER was much narrower when sample loading was conducted at 150 bar, the best peak shape was obtained when the sample was loaded at half the original pressure. Furthermore, the last method led to the highest peak areas attained for CCTKPESER (Figure S6A) and ATEEQLK (Figure S6B) out of all the column temperature methods. These observations indicate that improving the chromatography of hydrophilic peptides can require careful optimization. Another dynamic temperature method was developed to perform multiple temperature modifications on the analytical column in addition to the cooling and heating of the precolumn. In this method, a second round of thermal focusing was performed to encourage peptide retention and elution at the inlet of the analytical column during the gradient, while the middle of the analytical column was heated to 50 °C in another effort to improve chromatography (Figure S1C). Implementation of this dynamic temperature method resulted in well resolved peaks for CCTKPESER and ATEEQLK, with peak areas similar in magnitude to those produced by precolumn cooling alone (Figures S6A and S6B, respectively). Compared to the original precolumn cooling/heating method, the additional temperature changes were successful in restoring the loss of signal intensity experienced by these peptides. Finally, our observations on the improved chromatography of two BSA peptides obtained with constant precolumn cooling and the dynamic temperature methods show qualitative similarities with those achieved by Wilson et al., where cooling and heating of the sole analytical column improved the chromatography of early eluting BSA peptides.19

Ion-pairing chromatography is commonly used to improve the chromatography of poorly retained analytes. Acidic ion-pairing reagents can complex with the basic groups of peptides,35 thus increasing their hydrophobicity to promote retention to the stationary phase. We were interested in the effects of an ionpairing reagent (0.1% TFA) on the detection of early eluting BSA peptides. When the control temperature method was altered to load BSA samples in the presence of 0.1% TFA, two major differences were observed in comparison to the original control method. First, all of the monitored BSA peptides displayed up to a 5-fold reduction in peak area after being loaded onto the precolumn in 0.1% TFA (Figures S3A to S3E). It has been reported that the electrospray ionization signal loss associated with TFA use is typically in the range of an order of magnitude.36 The lower ion suppression we observed may be due to the absence of TFA in the buffers that compose the gradient. Second, the retention times of the TFA loaded peptides were considerably longer in many instances (Figures S4A to S4E), indicating that the ion-pairing reagent had the desired effect of increasing their hydrophobicity. In fact, no temperature method was capable of increasing the retention times of the peptides beyond those obtained with ion-pairing conditions. The presence of 0.1% TFA during sample loading produced resolved peaks for several early eluting peptides at retarded retention times (Figures 2A to 2C). While there were considerable increases in the peak areas of CCTKPESER (Figure 2D) and ATEEQLK (Figure 2E) because of TFA loading conditions, the increases were not as large as those obtained by precolumn cooling. Conversely, the ion-pairing reagent greatly improved the peak areas of SEIAHR (GRAVY score −0.95, Figure 2F) and four other BSA peptides (CASIQK, Figure S7A; DTHKSEIAHR, Figure S7B; NYQEAK, Figure S7C; and TPVSEK, Figure S7D), outperforming any of the other TFA-free temperature methods. When the TFA sample was run with the dynamic temperature method that performed multiple rounds of thermal focusing, no further increases in peak area were observed for the four aforementioned peptides. This demonstrates that the effect of TFA on the chromatography of these peptides was much larger than those obtained by our column temperature methods, but this was not unusual as the benefit from TFA is derived from the combination of ion-pairing and the added hydrophobicity from the trifluoroacetate ion itself.35 In summary, although more early eluting BSA peptides were resolved in TFA loading conditions, the majority of BSA peptides experienced a reduction in sensitivity due to ion suppression effects. Since proteomic analyses typically require as much sensitivity as possible, such a large compromise may not be acceptable. We also observed that the signal loss induced by TFA affected the charge states of CCTKPESER differently, with the triply charged precursor (Figure S6A) suppressed disproportionately more than the doubly charged precursor (Figure S6D). These phenomena do not occur in any of the TFA-free temperature methods, which indicate that while column temperature changes affect the peak intensities of CCTKPESER, they do not appear to alter its charge state distribution. Despite the appearance of fewer resolved BSA peptides near the beginning of the gradient, the absence of TFA-induced ion suppression and potential consequences on the charge states of peptides F


Technical Note

Journal of Proteome Research clearly highlights the LC−MS compatibility of the temperature methods. Although improvements in the chromatography of early eluting BSA peptides were demonstrated by several temperature methods, the overall BSA sequence coverage (Figure S8A), the number of peptides identified (Figure S8B) and the sum of peptide intensities (Figure S8C) were surprisingly unaffected. Intriguingly, only minor decreases in the sequence coverage and number of peptide identifications were reported for TFA loaded samples, despite the large decrease in total peptide intensity. It was discovered that CCTKPESER (Figure S9A), ATEEQLK (Figure S9B) and SEIAHR (Figure S9C) were identified by MaxQuant with respectable Andromeda scores, even in control runs where the chromatography of these peptides was comparatively poor. This suggests that these hydrophilic BSA peptides were present in quantities that could generate MS/MS spectra of acceptable quality, although we do not consider 50 fmol to be an excessive amount of sample for this study. Except for CCTKPESER, improvements in the chromatography of ATEEQLK and SEIAHR peptides did not always result in higher Andromeda scores, but it should be noted that such observations are likely to be influenced by the number of coeluting peptides and the stochastic nature of MS/ MS spectra collection by data-dependent acquisition. Although the BSA sample was useful for determining beneficial temperature methods, it became evident that it was inappropriate in both complexity and dynamic range to be used for in-depth proteomic studies. We therefore decided to investigate the effect of column temperature methods on a complex sample, with the expectation that column temperature and gradient changes should have a greater influence on the number of identified hydrophilic peptides.

Table 1. Summary of HeLa Peptide and Protein Identifications Obtained from Six Temperature Methodsa

a LC−MS runs containing 500 ng of sample were performed in duplicate.

We were interested in whether the temperature methods improved the chromatography of hydrophilic peptides. In order to objectively investigate the relative effects of each method, it was important to normalize all experimental runs due to the effect of temperature on retention times. Although peptides with variable modifications were present in samples at a very low percentage (Figure S10), there were concerns that large retention time differences could exist for peptides present in both the unmodified and modified form. We therefore excluded peptides with variable modifications from the analysis, with normalization performed by binning the average retention times of all the highest-scoring unmodified peptides from each method against the average retention times of four BSA contaminant peptides (Table S2), thus distributing all the average retention times into five time periods (Figure S11). To assist in the detection of hydrophilic peptides, the early eluting BSA peptide TCVADESHAGCEK was again used as the first reference point, which eluted approximately 25 min after the gradient began. The control method resulted in the identification of 411 unmodified peptides from the first time period (Table S3A), where a very high proportion of these were hydrophilic (391 peptides with GRAVY scores below zero). Although all column cooling methods produced a higher number of hydrophilic peptides identified from the first time period, this benefit did not necessarily apply to the final total of hydrophilic peptides. To illustrate this point, precolumn cooling alone resulted in the lowest total of hydrophilic peptides identified out of all the temperature methods (7052 peptides, Table S3D), despite the number of early eluting hydrophilic peptides actually increasing by 10% (431 peptides) over the control method. It is likely that many hydrophilic peptides were focused on the chilled precolumn, but that the same environment produces an inefficient elution that ultimately affects the number of hydrophilic peptides identified. The method that performed only cooling and heating of the precolumn produced a 40% increase in early eluting hydrophilic peptide identifications (546 peptides, Table S3C) in comparison to the control, indicating that the heating step promotes an efficient elution of hydrophilic peptides from the precolumn. Remarkably, 111 strongly hydrophilic peptides (GRAVY scores below −2) were

Effect of Column Temperatures on HeLa Samples

Since precolumn cooling improved the chromatography of several hydrophilic BSA peptides, we wanted to explore whether complex peptide samples could also benefit from column temperature changes. Tryptically digested HeLa samples (500 ng on-column) were analyzed by six temperature methods, with peptide elution conducted with 2 h gradients. We restricted the selection of temperature methods to those that demonstrated benefits for hydrophilic BSA peptides or possessed potential to improve the chromatography of hydrophilic peptides. Over 15000 unique peptide sequences were identified from individual runs with the control method at a 1% false discovery rate (Table 1), which also culminated in the detection of over 3000 protein groups. The overlap for duplicate runs was consistently above 80% at the peptide level and above 90% at the protein level for all temperature methods, indicating good reproducibility between the individual runs. The dynamic temperature method that specified chilling of the precolumn (during sample loading) followed by heating (during peptide elution) produced the best overall results, with the average number of peptide identifications increasing by approximately 15% and the average total of identified proteins up by 7%. Cooling of the precolumn alone resulted in the lowest identification totals of all methods, indicating that this single temperature change had a severe adverse effect, while no obvious benefits were produced by the remaining methods. It was unexpected that the dynamic temperature method involving two rounds of peptide focusing did not appear to be any better for the identification of HeLa peptides than the control method. G


Technical Note

Journal of Proteome Research identified from the earliest time period, which is an increase of approximately 76% over the control method (Figure 3A) and also the highest number obtained by any of the six methods (Tables S3A to S3F). Compared to the control results, narrower peak widths were generally observed for the population of hydrophilic peptides that eluted in the first time period (Figure S12). Finally, an unexpected side effect of this temperature method was the high number of unmodified peptides identified in the last time period (Table S3C). The dynamic temperature method that performed two rounds of thermal focusing resulted in the highest number of early eluting hydrophilic peptides (613 peptides), which was 57% better than the control method (Figure 3A). Further examination revealed similar sized increases in the number of early eluting peptides classified as weakly hydrophilic (214 GRAVY scores from less than 0 to −1) and moderately hydrophilic (312 GRAVY scores from less than −1 to −2). It was intriguing that these classes of hydrophilic peptides benefitted greatly from the additional temperature steps. The median peak width for the early eluting hydrophilic peptides was the smallest out of all the methods (Figure S12), although this result was not surprising since we showed this method to be advantageous for the chromatography of hydrophilic BSA peptides. Nonetheless, the different composition of GRAVY scores between the two most beneficial methods for early eluting hydrophilic peptides suggests that these methods exhibit peptide retention and/or elution biases based upon hydrophilicity. A method that only contained the analytical column temperature changes from the aforementioned dynamic thermal focusing method provided a 24% increase in the number of early eluting hydrophilic peptides over the control method (484 peptides), showing that an effective concentration and release of peptides can be performed on the analytical column (Table S3E). This HeLa method was formulated to examine the contribution of dynamic temperature changes performed on the analytical column in the absence of any precolumn temperature changes. Although many early eluting hydrophilic peptides were identified, this total was lower than those obtained by methods incorporating precolumn cooling and heating. Finally, when the middle section of the analytical column was heated to 50 °C, very few differences from the control results were observed (Table S3F). This outcome was largely expected due to the negligible length of time the peptides were exposed to heated conditions and the possibility that elevated temperatures can worsen the chromatography of hydrophilic peptides. This was aptly demonstrated by the early eluting hydrophilic peptides from this method possessing the highest median peak widths out of all methods (Figure S12). Out of the 14475 hydrophilic peptides identified from the control and two best performing dynamic temperature methods, approximately one-third could be detected by all three methods (Figure S13). For early eluting hydrophilic peptides, the number of identifications that could only be discovered by the dynamic temperature method (precolumn and analytical column thermal focusing) represented the largest group (Figure 3B), highlighting the ability of this method to identify unique peptides. Examination of the GRAVY scores from the three possible sets of uniquely detected peptides found that the noncontrol methods still exhibit the same hydrophilicity biases that were observed earlier, reinforcing the viewpoint that certain temperature methods can recover and identify exclusive groups of hydrophilic peptides (Figure 3C).

Figure 3. Dynamic temperature methods identify different populations of hydrophilic HeLa peptides. (A) GRAVY scores of early eluting hydrophilic peptides from the control and two dynamic temperature methods. (B) Overlap of early eluting hydrophilic peptides from the three HeLa temperature methods. (C) GRAVY scores of early eluting hydrophilic peptides uniquely identified by each column temperature method. (D) Retention lengths of 212 early eluting hydrophilic peptides commonly detected by the three HeLa temperature methods. In these box plots, the whiskers incorporate 1.5 times the interquartile range, while moderate and extreme outliers are denoted by open and closed circles, respectively. Two-tailed sign tests indicate statistically significant differences in the median values of these methods (p ≤ 0.001). H


Technical Note

Journal of Proteome Research One of the contributing reasons for these observations was the ability of both noncontrol methods to improve column efficiency, where the reduction in the median retention lengths of the 212 overlapping hydrophilic peptides (Figure 3D) was determined to be statistically significant between all three methods (two-tailed sign test, p ≤ 0.001). Effect of Column Temperatures on the Chromatography of Isoaspartic Acid Containing Peptides

In order to conduct an investigation into the effect of column temperatures on the separation of isoaspartylated peptides and their unmodified variants, pairs of coeluting isoaspartic and aspartic acid containing peptides were required. Peptide selection was based on previous studies demonstrating isoaspartic and aspartic acid containing peptides to be either coeluting or possessing similar retention times from long gradients.25,28 A mixture of 12 peptides was prepared, which comprises four distinct groups, with each group containing three peptides where an asparagine residue was substituted for an aspartic or isoaspartic acid (Table S1). We hypothesized that the aspartic and isoaspartic acid peptide pairs would coelute on a shorter gradient performed under control temperature conditions. However, only one of the peptide pairs coeluted in the middle of the gradient (peptides 12 and 11), while the remaining pairs eluted as fully resolved peaks at later retention times (Figure S14). It is difficult to establish why only one aspartic and isoaspartic acid peptide pair coeluted with our setup, although differences in chromatographical equipment among the isoaspartic acid peptide studies are likely to have contributed to the contrasting observations. Nonetheless, we manipulated the column temperatures to see if the coelution of the modified (AGFAGDDAPR, where D is an isoaspartic acid) and unmodified (AGFAGDDAPR) peptides could be altered. No improvement in the separation of these two peptides was detected when the temperature of the precolumn was modified or when heat was applied to any of the three temperature controlled areas, resulting in similar peak areas (Figures S15A and S15C) and retention times (Figures S15B and S15D). Although these peptides were weakly hydrophilic (GRAVY score −0.57), precolumn cooling did not increase the abundance of either peptide. Closer inspection of the chromatogram revealed peak shouldering with control column temperatures (Figure 4). However, cooling conducted at the inlet of the analytical column resulted in the appearance of two peaks, indicating that better selectivity was achieved with this method. We hypothesized that further chromatographical improvements could be obtained with additional chilling. When a long section from the middle of the analytical column was cooled, the difference in retention times increased and the peak shape of the earlier eluting peptide improved. Further improvements were obtained when both areas of the analytical column were simultaneously chilled, culminating in the best peak shape of the earlier eluting peptide and the largest separation of the isobaric peptides to date. This method also produced the highest discrimination score, which reflects the magnitude of the separation between the two peaks. The identity of both peaks was confirmed when equivalent amounts of the isoaspartic and aspartic acid containing peptides were run individually using the two best analytical column cooling methods (Figures S16A to S16D), with the isoaspartic acid variant eluting earlier than the unmodified form in both methods. In addition, the retention times from the single peptide runs were in good agreement with those obtained from

Figure 4. Effect of analytical column cooling on the elution of isoaspartic (AGFAGDDAPR) and aspartic (AGFAGDDAPR) acid containing peptides. Representative extracted ion chromatograms (m/ z 488.73, 2+) from the control temperature method (black), inlet cooling of the analytical column (light blue), middle section cooling of the analytical column (blue) and simultaneous cooling of both areas (dark blue) were overlaid. For the two latter methods, the identity of the peptide peaks (D or D) were assigned by comparing retention times with those obtained from single peptide runs. Discrimination factors that describe the degree of separation between adjacent peaks are given for each cooling method (inset).

the same peptides when they were part of the peptide mixture (Figures S15B and S15D). The method specifying both sections of the analytical column to be cooled provided the most accurate average peak area measurements of AGFAGDDAPR and AGFAGDDAPR, which were usually within 10% of the average peak area obtained from single peptide runs (Figures S15A and S15C). Unsurprisingly, peak quantification of both peptides from the mixture became more inaccurate as less of the analytical column was chilled, highlighting the importance of selectivity and peak shape for quantification purposes.

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CONCLUSION AND FUTURE PERSPECTIVES Several studies have previously demonstrated that the manipulation of column temperature allows improvements in the chromatography of small molecules14−16 and peptides.17−20 This report builds upon those findings by exploring the effect of different column temperatures on samples that are relevant to the proteomics field. Our finding that the chromatography of BSA hydrophilic peptides can be improved with precolumn cooling indicates that temperature alterations are a viable alternative to the use of ion-pairing reagents and other types of chromatographical media. The loading of peptides at subambient temperature should improve the proteomic characterization of analytically challenging samples (e.g., histones and neuropeptides) where many hydrophilic peptides are likely to be present. Techniques that heavily depend on peak shape, such as the quantification of hydrophilic peptides by targeted mass spectrometry approaches, could also benefit from chilled column temperatures during sample loading. Although precolumn cooling is important for the retention of hydrophilic peptides, subsequent precolumn heating helped to reduce peak widths and facilitate the efficient elution of HeLa peptides. Deeper proteome coverage of complex samples is therefore possible with the precolumn cooling/heating method. We observed differences in the ability to resolve hydrophilic peptides between the precolumn cooling/heating method and I


Technical Note


(http://www.proteomexchange.org) via the PRIDE partner repository37 with the data set identifier PXD006112.

the dynamic temperature method that performed multiple rounds of peptide focusing, with strongly hydrophilic peptides benefiting from the former method, while more weakly hydrophilic peptides were detected with the latter method. If the GRAVY scores from hydrophilic peptides of interest are known, one of these temperature methods could be preferentially selected to improve the chromatography of the peptides of interest, leading to more confident peptide identifications or greater sensitivity if quantification was the primary objective. Further investigations are necessary to explain the hydrophilicity bias displayed by the two methods, where the creation of a complex hydrophilic peptide mixture (where the GRAVY score distribution is also known) may assist in these studies. In conclusion, we show that column temperature can be modulated in a spatiotemporal manner to improve the chromatography of hydrophilic peptides, therefore extending the separation capabilities of reversed phase chromatography. The separation of an isoaspartic acid containing peptide with its unmodified isobaric version was possible with analytical column cooling. Hardware limitations prevented the possibility of observing further separation of the two peaks, specifically the inability of the DTF device to chill the entire analytical column, as well as the maximum backpressure limit being exceeded with colder column temperatures. This could be facilitated by redesigning the DTF apparatus so that the length of the third temperature controlled area covers more of the analytical column and performing the reversed phase chromatography on an ultrahigh-performance LC system, respectively. It would be interesting to investigate whether similar observations occur with a more complex mixture, especially for peptide pairs that elute early in the gradient. Such information would help determine which properties of an isoaspartylated peptide influences selectivity. Although the effect of column temperature on selectivity has been investigated, most of the attention has been focused on small molecules.11 It also remains to be established whether peptides containing other post-translational modifications could benefit from separations performed with subambient column temperatures. In conclusion, we have demonstrated the ability to improve the separation and detection of peptides by applying colder column temperatures at defined positions and relevant time points.

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ACKNOWLEDGMENTS We thank Ole Vorm (Evosep Biosystems) for making the DTF apparatus available for this study. The authors thank members of the Proteomics Program for constructive discussions. The work in this study was in part supported by The Novo Nordisk Foundation Center for Protein Research; The Novo Nordisk Foundation (grant numbers NNF14CC0001 and NNF13OC0006477); The Danish Council for Independent Research (grant numbers DFF 4002-00051 (Sapere Aude) and DFF 4183-00322A); and the Danish Cancer Society (grant number R146-A9159).

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(1) Aebersold, R.; Mann, M. Mass-spectrometric exploration of proteome structure and function. Nature 2016, 537, 347−355. (2) Wang, H.; Chang-Wong, T.; Tang, H.-Y.; Speicher, D. W. Comparison of extensive protein fractionation and repetitive LC−MS/ MS analyses on depth of analysis for complex proteomes. J. Proteome Res. 2010, 9, 1032−1040. (3) Di Palma, S.; Hennrich, M. L.; Heck, A. J.; Mohammed, S. Recent advances in peptide separation by multidimensional liquid chromatography for proteome analysis. J. Proteomics 2012, 75, 3791−3813. (4) Washburn, M. P.; Wolters, D.; Yates, J. R., III. Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat. Biotechnol. 2001, 19, 242−247. (5) Xie, F.; Smith, R. D.; Shen, Y. Advanced proteomic liquid chromatography. J. Chromatogr. A 2012, 1261, 78−90. (6) Iwasaki, M.; Miwa, S.; Ikegami, T.; Tomita, M.; Tanaka, N.; Ishihama, Y. One-dimensional capillary liquid chromatographic separation coupled with tandem mass spectrometry unveils the Escherichia coli proteome on a microarray scale. Anal. Chem. 2010, 82, 2616−2620. (7) Köcher, T.; Swart, R.; Mechtler, K. Ultra-high-pressure RPLC hyphenated to an LTQ-Orbitrap Velos reveals a linear relation between peak capacity and number of identified peptides. Anal. Chem. 2011, 83, 2699−2704. (8) Liu, H.; Finch, J. W.; Lavallee, M. J.; Collamati, R. A.; Benevides, C. C.; Gebler, J. C. Effects of column length, particle size, gradient length and flow rate on peak capacity of nano-scale liquid chromatography for peptide separations. J. Chromatogr. A 2007, 1147, 30−36. (9) Jorgenson, J. W. Capillary liquid chromatography at ultrahigh pressures. Annu. Rev. Anal. Chem. 2010, 3, 129−150. (10) Ma, L.; Carr, P. W. Loss of bonded phase in reversed-phase liquid chromatography in acidic eluents and practical ways to improve column stability. Anal. Chem. 2007, 79, 4681−4686. (11) Dolan, J. W. Temperature selectivity in reversed-phase high performance liquid chromatography. J. Chromatogr. A 2002, 965, 195− 205. (12) Antia, F. D.; Horváth, C. High-performance liquid chromatography at elevated temperatures: examination of conditions for the rapid separation of large molecules. J. Chromatogr. 1988, 435, 1−15. (13) Vanhoenacker, G.; Sandra, P. Elevated temperature and temperature programming in conventional liquid chromatography fundamentals and applications. J. Sep. Sci. 2006, 29, 1822−1835. (14) Holm, A.; Molander, P.; Lundanes, E.; Greibrokk, T. Novel column oven concept for cold spot large volume sample enrichment in high throughput temperature gradient capillary liquid chromatography. J. Sep. Sci. 2003, 26, 1147−1153. (15) Groskreutz, S. R.; Weber, S. G. Temperature-assisted on-column solute focusing: A general method to reduce pre-column dispersion in capillary high performance liquid chromatography. J. Chromatogr. A 2014, 1354, 65−74.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jproteome.6b01055. Figures S1−S16; Tables S1−S3 (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*Tel: +45 35 33 06 21. E-mail: cliff[email protected]. *Tel: +45 35 32 50 19. E-mail: [email protected]. ORCID

Clifford Young: 0000-0002-3945-286X Notes

The authors declare no competing financial interest. Mass spectrometry raw data and MaxQuant search output files have been deposited to the ProteomeXchange Consortium J


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Improved Reversed Phase Chromatography of Hydrophilic Peptides

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