Nano-LC in proteomics: recent advances and ...

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Nano-LC in proteomics: recent advances and approaches

In proteomics, nano-LC is arguably the most common tool for separating peptides/ proteins prior to MS. The main advantage of nano-LC is enhanced sensitivity, as compounds enter the MS in more concentrated bands. This is particularly relevant for determining low abundant compounds in limited samples. Nano-LC columns can produce peak capacities of 1000 or more, and very narrow columns can be used to perform proteomics of 1000 cells or less. Also, nano-LC can be coupled with online add-ons such as selective trap columns or enzymatic reactors, for faster and more automated analysis. Nano-LC is today an established tool for research laboratories; but can nano-LC-based systems soon be ready for more routine settings, such as in clinics?

Nano-LC combined with electrospray ionization (ESI)–MS is the most commonly used analysis platform for advanced proteomics. Nano-LC columns can be defined as having inner diameters (IDs) of 0.1 mm or lower [1] and are operated at nl/min flow rates. Capillary format columns, for example, 100–300 Pm ID are also used in proteomics, but to a lesser extent. In ‘bottom-up’ proteomics, the role of the nano-LC system (in this context) is to physically separate peptides (of enzymatically cleaved proteins) from each other with high resolution. The peptides enter the ESI–MS at different time points, with the chromatographic separation process lasting from 30 min to tens of hours [2,3] . This allows the ESI–MS to process/detect a large number of peptides, as ion suppression effects [4] and scan rate limitations are minimized. Short run times may be sufficient for handling less complex mixtures (e.g., extracts from immunoprecipitations), while long run times may be used to maximize output for comprehensive analysis of, for example, an organism. As with larger LC columns, nano-LC columns typically separate compounds according to hydrophobicity (reversed phase LC [RPLC]), but other separation principles are also used in today’s nano-LC-based proteomics.

10.4155/BIO.15.92 © 2015 Future Science Ltd

Steven Ray Wilson*,1, Tore Vehus1, Henriette Sjaanes Berg1 & Elsa Lundanes1 1 Department of Chemistry, University of Oslo, Post Box 1033, Blindern, NO-0315 Oslo, Norway *Author for correspondence: Tel.: +47 97010953 [email protected]

The path to nano-LC for MS-based proteomics began decades ago; in 1980, Takeuchi and Iishi began packing ‘ultramicro’ columns with diameters as low as 0.1 mm [5] ; a key motivation was to prepare columns with flow rates that would be compatible with MS instrumentation. During the 1980s, Hirata and Jinno [6] , and Karlsson and Novotny [7] and others began to pack efficient nano-LC columns (down to a40 Pm ID), with a motivation of performing analysis of limited amounts of sample. At the end of the decade, Lee et al. coupled microbore LC (1 mm ID) and the newly established ESI–MS, demonstrating this hyphenation with tryptic peptides of recombinant bovine somatotropin [8] . In the early 1990s, Henderson et al. performed endogenous peptide identifications by coupling narrower 100 Pm ID LC columns to ESI–MS [9] . In 1996, Chervet and Ursem layed a framework for much of today’s nano-LC instrumental practices [10] . Logically coupled with nanoelectrospray (suited for nl/min flow rates) [11] , the now fairly well-developed nano-LC was fully compatible with MS analysis and became an established approach for proteomics in the new millennia [12] .

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Wilson, Vehus, Berg & Lundanes

Key terms Downscaling: Downscaling formula [inner diameter column 1/inner diameter column 2] 2 tells the potential gain in both sensitivity (when injecting the same amount) and reduction of flow rate, when ID 1 > ID 2. Peak capacity: Peak capacity formula [retention time last peak – retention time first peak]/average peak width tells how many compounds a column can separate.

Today, nano-LC columns can physically separate 1000 or more compounds in a single operation [13] , and in combination with today’s high performance mass spectrometers, around 5000 proteins can be identified in a single recording of a sample [14] . Although these are impressive feats, proteomics-based research continues to have challenges regarding ‘Proteome coverage’: there are tens/hundred thousands of protein species encoded for in humans [15] , and virtually endless possibilities/combinations of proteoforms [16] ; ‘automation’: although the LC and ESI–MS are elegantly hyphenated, preceding sample preparation steps can be very manual and even difficult; ‘robustness’: for nanoLC–MS to become more attractive for clinical applications, the technology needs to convince regarding, for example, repeatable performance in routine settings, and be reproducible for other laboratories. In this review, we will present contemporary concepts and technological advances in nano-LC (such as novel columns and online systems), and so discuss whether these can be used to meet the three challenges described above. Advantages (& misunderstandings) of nano-LC The main advantage of nano/capillary LC is enhanced sensitivity, allowing for ultratrace detection of limited sample amounts [17–19] . The enhanced sensitivity is due to the reduced radial dilution of chromatographic bands in columns with narrow IDs (Figure 1) [20,21] . Radial dilution is proportional with the square of the column’s radius, so, for example, a chromatographic band traveling through a 0.05 mm ID column will, according to the downscaling factor, be diluted almost 2000 times less than when using a 2.1 mm ID column of the same length, etc. This factor also represents the theoretical gain in sensitivity for an amount injected, when using a concentration-sensitive detection platform (such as ESI–MS). The sensitivity associated with downscaling has been illustrated by, for example, Shen et al., who showed remarkable increases in signal intensity when injecting the same sample on columns from 75 to 15 Pm ID [22] . It should however be noted that larger amounts of sample can be injected on conventional columns, largely compensating for its

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larger radial dilution. Thus, the key advantage of nanoLC is ‘making the most of’ limited samples (e.g., one drop of blood vs access to tens of milliliters of blood). A potential misunderstanding is that a narrow ID has inherently higher chromatographic efficiency and resolution (and hence peak capacity). However, the relationship between ID and column performance is not straightforward. For instance, ultrahigh pressure LC (UHPLC) columns perform best in narrow ID format because resolution-damaging radial temperature gradients (arising from friction effects due to high backpressures) become less prominent [11] . Also, monolithic columns [12,13] typically perform better in nano/capillary format due to easier in-column polymerization [14] . However, core shell particles [15] have been more difficult to pack in narrow columns compared with, for example, 4.6 mm ID columns and have traditionally shown best performance in conventional LC format [16] . See below for more details on the above-mentioned column types. Another potential misunderstanding is that nanoLC is slower and gives higher backpressures. However, narrow columns typically operate optimally at the same linear velocities (cm/s) as larger bore columns, even though the volumetric flow rate is much lower. Therefore, the backpressure and time of separation are typically comparable regardless of column ID. Indeed, nano/capillary LC often has longer sample analysis cycles, but this is mostly due to analyte enrichment steps often required (see discussion below). The low flow rates of nano/capillary LC are also advantageous regarding the ESI mechanism. The ESI unit generates smaller droplets at reduced flow rates, which leads to improved transfer of ions into the MS [23] . At very low flow rates (low nl/min), an additional feature is diminished ion suppression [23,24] , which allows for significantly enhanced sensitivity. Other advantages include reduced consumption of solvents, which can be especially important if expensive reagents are to be used, and responsiveness to temperature [25] , which can be used to fine-tune separations, although this approach is rarely used for peptides/proteins. Plug & play? Commercial nano-LC columns Today, a considerable number of nano-LC columns and LC-systems are commercially available (see Tables 1 & 2). Typically, the columns are 50–75 Pm ID; however, smaller ID columns are also available from, for example, Unimicro (25 Pm ID). Column lengths are typically 150 mm, with longer columns being more suited for high-peak capacity comprehensive proteomics, and shorter columns being more suited for, for example, faster, targeted analysis. Most of the columns are packed

future science group

Nano-LC in proteomics: recent advances & approaches

Review

Peptide mixture

Radial dilution

Diluted band enters detector: = weaker signal Same axial dilution

Reduced radial dilution

More concentrated band enters detector: = stronger signal

Figure 1. Chromatographic dilution processes with a conventional and a narrow column. Radial dilution is a function of the square of the column radius. Therefore, when using a narrow ID, compounds enter the ESI–MS in higher concentrations, resulting in enhanced signal. Axial dilution of chromatographed compounds (e.g., peptides or proteins) is typically independent of ID.

with silica particles with C18 chains covalently attached (the stationary phase; Figure 2C). An increasing number of particles are of smaller size (1.4–2 Pm), which allows for increased chromatographic efficiency but at the cost of higher backpressure (UHPLC). Some columns are however packed with core shell particles (typically larger than 2 Pm), which have comparable efficiencies to UHPLC but at lower backpressures, however possibly with lower loading capacities (due to reduced surface area). A far more limited number of commercial columns are made of single piece monoliths, synthesized within a capillary (Figure 2B) [26–29] . A common feature of monolithic columns is a vastly lower backpressure compared with packed columns. The most ‘plug and play’ variants of nano-LC are arguably the chip format columns by, for example, Agilent [30] with integrated online SPE for enrichment (see more details/pros and cons of online systems below). Also, Thermo provides simply installed columns/emitters with integrated heating jackets [31] (see discussion on effect of temperature on nano-LC columns below). One recent comparison between nano-chip column and classical columns for protein identification concluded that more proteins could be identified with the former [32] . In contrast to most analytical chemistry fields, many proteomics/nano-LC researchers pack or synthesize their own columns.


Do it yourself: self-packed nano-LC columns Self-packing of nano-LC columns tends to be cheaper than purchasing commercially available columns and increases flexibility regarding choice of stationary phases and particle size. Columns are typically packed in fused silica capillaries, as is typically the case for commercial columns as well. Packing techniques traditionally include slurry-packing (packing with a liquid), dry packing (packing with gases like N2, H2 or Ar [35]) or packing with supercritical fluid CO2 [36–39] . A major challenge is to obtain a uniformly packed bed (a key basis for an efficient column) especially with regard to small particle sizes, narrow column IDs or long columns [37,40–41] , which is typical in today’s proteomics. Effects of, for example, temperature, packing pressure, slurry- and packing liquid, and concentration have been studied in detail (also) in recent studies [41–48] . However, ‘rules of thumb’ are difficult to establish regarding optimal packing conditions for capillary/nano columns and materials. Capriotti and Leonardis et al. showed that particle sedimentation (associated with column clogging and poor efficiency) decreases with higher temperature [36,43] ; columns packed at 70°C were packed faster and gave significantly lower plate heights for all slurries tested, compared with the columns packed at room temperature. On the other hand, empirical data by, for

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Table 1. Nano-LC columns for peptide separations. Name

ID (Pm)

Lengths (mm)

Available phases Particle/pore size Company

InetrSustainSwift

100

50, 150, 250

C18

3 Pm

GL Sciences

Comment

PepMap™ precolumn 75

20

C18

3 Pm

Thermo Fischer Scientific

PepMap™

75

50, 150, 250, 500

C18

3 Pm/100 Å

50

150

C18

Accucore™

75

150

C18

ProSwift™

100

500

Phenyl

PepSwift™

100

50, 250

PS-DVB

Monolith

Dionex

100

50

C18

Silica monolith

Merck-Millipore

50

150

C18

100, 150, 250

C18

1.8 Pm/100 Å

Waters

75 50

Chromolith

®

HSS T3 nanoACQUITY 75

2.6 Pm/100 Å

Solid core

100

100

C18

75

100, 150, 250

C18

1.7 Pm/130 Å, 300 Å

100

100

C18

1.7 Pm/300 Å

Peptide CSH C18 nanoACQUITY

75

100, 150, 200

C18

1.7 Pm/130 Å

ChromXP-C18/C8-CL

75

50, 150

C18/C8

3, 5 Pm/120 Å, 300 Å

Chrom-XP-C4

75

50, 150

C4

3, 5 Pm/120 Å, 300 Å

Proteins

HALO® C18/C8

75

50, 150

C8/C18

2.7 Pm

Solid core

HALO® HILIC

75

50, 150

HILIC

2.7 Pm

Solid core

Dr. Maisch Reprosil C18

75

50, 150

C18

1.9–10 Pm

Ascentis® Express

100

50, 150

C18

2.7 Pm

Sigma-Aldrich

75

50, 150

C18

50

200, 250, 300, 350, 400

C4 / C8 / C18

C18, C8, 3 Pm

Unimicro technologies

Peptide BEH C18 nanoACQUITY

Unimicro technologies

75

Proteins and peptides

Solid core

C18, C8, 3, 5 Pm C18, 3, 5 Pm

100 50

Eksigent (AB SCIEX) Eksigent

C18

C18 NPS, 1.5 Pm

C30

1.5 Pm NPS

Phenyl

3, 5 Pm

Solid core

75 100 50 75 100 50 75 100 BEH: Ethylene bridged hybrid; CSH: Charged surface hybrid; ID: Inner diameter; HILIC: Hydrophilic interaction LC; HSS: High strength silica; PS-DVB: Poly(styrene-co-divinylbenzene); NPS: Non-porous silica.

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Table 1. Nano-LC columns for peptide separations (cont.). Name

ID (Pm)

Lengths (mm)

Available phases Particle/pore size Company

Biosphere

25

50, 100, 150, 200

C18

3 Pm

NanoSeparations

9 bar/cm

50, 100, 150, 200

C18

1.9 Pm

NanoSeparations

17 bar/cm

50

Comment

75 100 25 50 75 100 SeQuant™ ZIC -HILIC 75

150

Zwitterionic HILIC 5 Pm/200 Å

EMD Millipore

PicoFrit

150

C18

5 Pm/300 Å

New Objective

®

75

BEH: Ethylene bridged hybrid; CSH: Charged surface hybrid; ID: Inner diameter; HILIC: Hydrophilic interaction LC; HSS: High strength silica; PS-DVB: Poly(styrene-co-divinylbenzene); NPS: Non-porous silica.

example, Shelly et al. [46] and Blue and Jorgenson [42] , and a mathematical simulation by Vissers et al. [48] indicate that a slow packing velocity is instead beneficial. In some studies [36,41,43] , the packing was performed with a conventional HPLC pump and resulted in columns providing reasonable peak capacity, demonstrating that it is not an absolute requirement to pack columns with thousands of bars as done by others [42,44–45] , even though high pressure is beneficial [42] . Leonardis and Capriotti et al. found water: isopropanol (10:90) mixture as slurry liquid and water:acetonitrile (10:90) mixture as packing liquid suitable for reversed phase materials [36,43] . However, it should be kept in mind that optimal slurry conditions will greatly depend on the particles/stationary phase to be packed. Regarding slurry-concentration (particles/ml liquid), optimal concentration can depend on column ID, slurry-liquid, and particle type [40] . Bruns et al. (0.9–1.9 Pm, both fully porous and core shell particles) and Blue and Jorgenson (1.1–2.7 Pm, both fully porous and core shell particles) found that higher slurry concentrations tended to give better column efficiencies [40,42] , tested with column IDs of 30–75 Pm, packed with pressures of 200 bar and up to approximately 2000 bar. In contrast, Kennedy and Jorgenson (5 Pm particles) were not able to pack very narrow columns (2.7 Pm fused-core C18 packed column > silica-based C18 monolith > PS-DVB monolith) [74] . Open tubular nano-LC for very limited samples Open tubular columns differ from other nano-LC columns as they are not filled with a separation medium, but have a relatively thin polymer layer attached to the walls of the capillary (polymer layer open tubular [PLOT] columns; [75]). PLOT columns have an ID of 20 Pm or less (Figure 2A) and are typically 1–10 m long. They have excellent separation properties, low carryover, and their manufacturing is simple and reproducible [33,76] . In a recent review, various approaches to enhance the surface area of stationary phases (PLOT) format are addressed [77] . Open tubular columns for LC have been described since the late seventies [78] , but Karger et al. were the first to show the potential of PLOT columns for proteomics [76] , using a 10 Pm ID column with a A

PS/DVB polymer layer. Since then, such columns have been employed for both targeted and comprehensive proteomics of limited samples, for example, laser micro dissection tissues and small numbers of cancer cells [18,79–80] . PLOT columns are particularly suited for analysis of very limited samples, as they have exceptionally low radial dilution. Today, PLOT LC can be operated with commercial instrumentation [81] . However, a bottleneck for the full exploitation of the resolving power of PLOT LC columns is a lack of commercial products (T-pieces, etc.) dedicated for low nl/min flow rates (PLOT columns are operated at 5–20 nl/min). This may be a source of void volumes, which limits the achievable PLOT LC resolution. Also, today’s nanoLC pumps cannot repeatably perform solvent gradients with such low flow rates, so a split plumbing must be employed with PLOT LC (set prior to the column, so no sample is lost), with a split ratio that must be checked and adjusted manually. As of today, PLOT columns are not yet commercially available. Enhancing nano-LC performance with solvents & temperature In addition to the column’s dimensions/traits, the mobile phase can also be a significant factor regarding peak capacity and sensitivity. For example, mobile phases that contain high amounts of an organic modifier (e.g., MeOH or ACN) are often associated with enhanced sensitivity, due to improved electrospray efficiency [82] . It has also been shown that nano-RPLC–MS sensitivity can be enhanced 10-fold by, for example, simply adding small amounts of DMSO to the mobile phase; however, the effect is MS instrument-dependent [83] . Hydrophilic interaction LC (HILIC) is an alternative to RP chromatography which is well suited not

B

Det HV Spot WD Mag LFD 10.0 kv 40 10.5 mm 15000x

10 μm

10 μm ID PS/DVB-PLOT

Review

C

50 μm

100 μm ID silica monolith

50 μm ID particle packed

Figure 2. Column morphologies. (A) 10 Pm ID PLOT LC column, (B) a 100 Pm ID silica monolith and (C) a 50 Pm ID solid core particle (2.6 Pm, Thermo Fisher Scientific) in-house packed column. PLOT: Polymer layer open tubular. Reproduced with permission from [33,34] © Elsevier (2010, 2011).



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only for, for example, small polar compounds but also for peptides [84] . HILIC mobile phases contain high amounts of ACN (95–45%). Its retention mechanism is made up of partitioning between an immobilized water layer in the stationary phase (which are typically polar/charged functional groups) and the mobile phase, as well as electrostatic and normal phase interactions, amongst others [84] . Its efficiency is often comparable to RP, and Horie et al. have shown that nano-HILIC silica monoliths are highly suited for comprehensive proteomics, enjoying a fivefold average increase in sensitivity compared with RPLC [58] , likely due to the high ACN content in the mobile phase. Malerod et al. have recently found that similar columns were very suited for glycomics, and a HILIC silica monolith was successful in retaining/separating polar glycosylated peptides for analysis with MS [61] . A HILIC methacrylate based monolithic column also showed great potential for peptide separation [85] . Palma et al. found HILIC to provide excellent sensitivity using packed columns [86] . HILIC is gaining ground in many application areas (from extremely polar compounds to lipids), but is still relatively new compared with RP, and is for the moment considered to be less robust and requiring higher maintenance. For example, the wrong choice of injection solvent significantly distorts the chromatography [87] , and column-washing maintenance procedures can be timetaking. Also, HILIC is very commercially limited in nano-LC format. Increased temperature reduce the viscosity of the mobile phase, and can therefore allow the use of longer nano-columns or higher flow rates due to lowered backpressures. Changing temperature can also be beneficial chromatography-wise, as increased temperature can reduce band broadening effects at non-ideal (efficiency-wise) flow rates [88] . At ideal flow rates, increased temperature is of less importance for enhancing efficiency for peptides (or smaller molecules) than for intact proteins, as temperature increase significantly increases the diffusion rates of these large molecules in and out of the stationary phase and pores. However, Hyung et al. have shown that column temperature elevation improves the efficiency and repeatability of peptide separations on 75 Pm ID columns longer than 70 cm (packed with 3 Pm particles) up to 60°C [89] , and Rogeberg et al. have shown that a temperature of 80° gave the highest peak capacity per time unit for peptides on 100 Pm ID RP silica based monolithic columns [34] . Increased temperature can however also be disadvantageous; in some cases, a significantly reduced ESI–MS signal has been associated with temperatures above 40°C degrees [33] although the reason for this is not established.

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Eghbali et al. have investigating the possibilities of ‘freezing’ the outlet of separation columns to refocus axially diluted chromatographic band [90] , and Groskreutz et al. recently published a study on oncolumn focusing by reducing the column temperature to 5°C prior to the separation (performed at 60°C), which improved peak widths and sensitivity [91] . Such approaches are highly compatible with nano-LC columns, as their narrow ID and fused silica column body material are very responsive to temperature. In addition to aiding focusing, temperature can be used to enhance selectivity of targeted analytes, by, for example, temperature pulsing (i.e., rapid changes in temperature) [92] . This approach has so far been demonstrated with small molecules, but could also be effective for peptides as well. SPE-nano-LC: online (selective) enrichment With conventional LC, volumes of, for example, 5 Pl or more are injected directly on to the column without time or efficiency issues. To enable injection of similar volumes using nano-LC, an online SPE step is usually performed [93] , using a trap column. This enriches the proteins/peptides prior to nano-LC and also functions as a desalting step. However, depending on the sample, an additional off-line SPE step may be practical to remove very hydrophobic compounds (e.g., sterols and phospholipids) that can irreversibly adsorb to the trap column and other parts of the nano-LC system [94] . For simple RP-based enrichment, commercial trap columns typically contain the same particles/material as the analytical column. However, it can be beneficial to employ a trap column with a relatively lower hydrophobicity compared with the analytical column. This allows for peptides/proteins to be refocused at the beginning of the analytical column after elution from the trap column, resulting in improved chromatography. Most important is however to not use a trap column with a higher hydrophobicity than the LC column, as this can be a major cause of poor performance. Exigent Technologies launched the first commercial nano-LC system (with trap column functionality) in 2004, and today most major commercial LC system manufacturers provide nano-LC instrumentation (Table 2) . Differences between the systems include pressure limits, column format (chip or non-chip) and add-ons such as temperature control (see Table 2). Some systems perform the SPE trapping with the same solvent pump that performs the LC gradient (e.g., the Thermo Easy-nLC system), while other systems have a dedicated pump for the SPE step [93] , including the Agilent CHiP system. Regarding the two-pump approach, an advantage is that solvent flows through



the LC column during all stages (e.g., loading, SPE conditioning). This is not possible with the more common, compact one-pump set-up, and additional time is spent on flow/pressure stabilization steps. It should be mentioned that chip-based systems for proteomics also exist for capillary dimensions, for example, the Waters ionKey™ (150 Pm column). In addition, other chip systems exist for complementary analyses, for example, glycomics [95] . More selective online trapping can be performed, for example, phosphopeptide isolation (using immobilized metal affinity columns) [96] , antibodies/antipeptides (specific proteins or peptides) [97,98] , or glycopeptide trapping (using, for example, boronate materials [99]). 2D nano-LC: off-line or online? To maximize information output in comprehensive proteomics, two orthogonal (i.e., unrelated) separation steps can be performed in series, for example, first chromatographing the sample according to isoelectric point, and so separate the peptides/proteins by their hydrophobicity [100] . Such approaches are called 2D chromatography [101] , and are analogous to 2D gel electrophoresis. 2D LC is far more efficient for increasing resolution than only expanding the gradient or column length in 1D LC (an approach with limitations [49]). For nano-LC, online 2D trap columns are commercially available, consisting of an ion-exchanger (IEX) material and a RP material placed in series [102] . Peptides are first trapped on the IEX section, and low charged (az = 1) peptides are so eluted on to the RP section with a moderate ionic strength solution (and are hence separated from the higher charged species). Subsequently, RPLC separation of this fraction is performed (separating the low charged peptides from each other, based on their hydrophobicity). Moderately (az = 2) charged peptides are then flushed onto the RP section with a solution of higher ionic strength, etc. Such approaches can provide improved proteome coverage compared with using a single LC separation [103] . However, comprehensive online 2D LC has not reached the same level of maturity as its sibling 2D GC (GC × GC), which is, for example, well suited for metabolomics [104] . A core reason is the limited possibilities for simple combination of LC separation principles in a single online system. For example, HILIC mobile phases (high ACN amounts) must not enter the RP column (requires low to moderate amounts of ACN to separate peptides), so online HILIC-RP can require rather intricate technical solutions; for example, trap columns may be placed between the two dimensions, to isolate analytes from the 1D mobile phase prior to separation in the 2D [105,106] . Between-


Review

Key term Orthogonality: In chromatography, a high orthogonality between two columns means there is little overlap in their separation mechanisms, and the columns can be combined in 2D LC systems for increased peak capacity (e.g., HILIC/SCX with RP).

dimension trap column setups can also be used for IEX-RP to allow favorable 1D conditions [107] . A more simple approach, off-line 2D LC is often used in proteomics. An common approach is to first separate peptides with a 4.6 mm RP column (to load larger amounts of sample) using a basic mobile phase, collect fractions off-line, and chromatograph these with a nano RPLC column using an acidic mobile phase [108] . Acidic-RPLC and basic-RPLC have surprisingly high degree of orthogonality [101] , and the off-line combination of these could be used to identify over 8000 proteins in a sample [108] . 3D LC (ERLIC, RPbasic and RPacid) for plasma proteomics has also been described [109] . Such an approach has the potential for providing high resolution, but is arguably very difficult to set up online. An online RPbasic × RPacid LC system has recently been described by the Mondello group (using capillary LC columns) [110] . Immobilized enzymatic reactors coupled with nano-LC Time-wise, a bottleneck of proteomics is sample preparation. For example, enzymatic digestion of proteins to ‘MS-friendly’ peptides can require hours of reaction time. Immobilized enzymatic reactors (IMERs) in column format [111] for rapid digestion (seconds to minutes) of proteins into peptides have been under development for many years. However, few papers have reported demonstrations with complex biological samples or implementation in online systems (which would allow fast and automated handling of small samples). Recently, Hustoft et al. have prepared IMERs in open tubular format (open tubular enzyme reactor [OTER]), with trypsin and lys C attached to the column walls [19,81] . These OTERs were online coupled with PLOT LC, and the OTER–PLOT LC–MS system could be used to detect proteotypic peptides of trace level proteins in extract from just 1000 cells, performing digestion of complex samples + separation in less than 2 h [19] (Figure 3) . Yuan et al. have recently reported a novel organic-silica monolithic based IMER (trypsin), which was also online integrated with a nano-LC system, allowing for over 3000 proteins to be identified from 10 Pg protein extract [112] . Importantly, the carry-over was remarkable low (as is the case with the OTER), which is a major factor for routine analysis. Wang et al.


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Injected proteins

Peptides transferred to trap column

To LC

Pre-column Waste

V1

V2

plit

W

Lo

ad

s nt-

ing

die

-w as

a

Gr

PLOT-column

te

OTER

100

AXIN1 proteotypic peptide

2 h, 1000 cell targeted proteomics

80 60 40 20 0 0

10

20

30

40

50

60

Time (min) Figure 3. Open tubular enzyme reactor coupled online with a very narrow, polymer layer open tubular nano-LC column. Between the OTER and the PLOT columns is an SPE trap column for enrichment and desalting. The fully automated system could be used to detect trace amounts of AXIN1 in just 1000 cells. OTER: Open tubular enzyme reactor; PLOT: Polymer layer open tubular. Adapted from [19] © Hustoft et al. (2014), under CC-BY license.

have developed an online digestion-nano-LC platform [113] ; the team demonstrated that stable isotopic labelling corrects for incomplete digestion on enzymatic reactors, allowing for accurate quantifications in cell lines [113] . Expecting more: is nano-LC ready for clinics? In medical settings, conventional LC is commonly used for measuring metabolites of diagnostic value, and also peptides and proteins. However, even though it can provide high sensitivity and can feature many automated sample preparation add-ons, nano-LC is still remains on the sideline in clinical analysis. To be an attractive option for clinics, nano-LC has to exhibit some of the same traits as more conventional systems (e.g., high-throughput analysis, robustness, ease of operation). High-throughput analysis is one of the most important aspects of clinical analysis, as time is an important factor in diagnosis as is also cost-efficiency. For instance, the introduction of UHPLC (typically 1 mm column ID systems) has reduced analysis time significantly in many application areas. However, a

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loss in speed is considered a major drawback with miniaturized systems, as in many of today’s commercial systems much time is spent on conditioning trap and analytical column, and stabilizing flow rates prior to analysis. Another undesirable trait of miniaturized systems such as nano-LC is reduced robustness/ease of operation. Column-housing and capillary connections etc. are more fragile in such systems and puts larger demands upon the operator. However, introduction of new column connections (e.g., NanoViper™ from Thermo or the nanoAcquity connections from Waters) may increase the robustness. Despite these factors, examples of nano-LC in the clinics exist, and Table 3 lists some representative studies with successful applications. Encouragingly, a recent study on cancer relevant proteins by Abbatiello et.al showed that reproducible quantitative results could be obtained between labs on nano-LC systems [114] . In Table 4, we summarize the performance of various approaches we have described in this review, regarding a number of factors appreciated in, for example, clinical settings.



Conclusion & future perspective Key factors that nano-LC can potentially contribute to increasing proteome coverage with are increased peak capacities with materials very suited for narrow IDs and, enhanced sensitivity to enable detection of ultralow abundant proteins. Regarding the first key factor, today, very long columns can be used for deep proteome mining; but can the peak capacity of nanoLC be pushed very much further? For instance, the silica-based monoliths efficiency has remained more or less the same since its introduction. The performance of packed columns may be pushed further by reducing the particle size (e.g., 1 Pm and lower), but such columns are as of today difficult to prepare for optimal performance [42] . Perhaps alternative approaches to packed or monolithic columns may be redemptive, for example highly ordered pillar array systems [115] . In addition, 2D LC (or maybe even 3D LC) has the potential for dramatically increasing output. However, novel separation materials may be required to further enhance total peak capacity and orthogonality in 2D LC. In addition, the pro-

Review

teomics community must not forget the importance of chemometry for optimizing, for example, fraction frequencies and flow rates when developing 2D LC approaches [116] . Regarding the second key factor, enhanced sensitivity can be obtained by, for example, further narrowing the ID of the nano-column (e.g., PLOT columns). However, to be able to not only detect ultratrace proteins, such columns must also be able to handle a certain dynamic range without being overloaded. Hence, increasing the loading capacity of nano-LC columns can be a valuable research area. Online add-ons (e.g., enrichment columns, heartcutting and selective trapping columns) are increasingly commercially available and popular. Therefore, it is expected that online solutions, which are often very compatible with nano-LC, will increasingly replace very manual approaches such as in-gel digestion procedures. Also, it is only a matter of time before comprehensive 2D LC systems for nano-LC become more available, given its great potential. Thus, it can be expected that nano-LC/proteomics will enjoy a

Table 3. Recent clinical relevant studies using nano-LC. Target/study

Sample

NanoColumn

Analysis length

LOD/LOQ US FDA validated?

Additional

Year

Ref.

GMA2-activator Urine protein lung cancer biomarker

150 Pm × 30 mm (5 Pm, 200Å) + 75 Pm X 200 mm (3 Pm, 200 Å)

N/A

N/A

Glycosylation

2013

[111]

GIP1–42 and GIP3–42 Plasma

PepMap 300 Pm × 1 mm (5 Pm) + PepMap 75 Pm × 150 mm (3 Pm)

>35 min

1 pM Full method GIP1–42 10 validation, no pM GIP3–42 comparison

2013

[112]

2 h

N/A

N/A

Total proteome 2008

[115]

75 Pm × 65 cm (3 Pm)

>2 h

N/A

N/A

Samples 2011 prefractionated

[116]

75 Pm × 50 mm (2 Pm, EasySpray)

4h

N/A

N/A

2015

[117]

Target protein(s)

Prostate specific antigen

Plasma depleted Trap + 75 Pm × of albumin and 150 mm (5 Pm, IgG 300Å)

48 plasma proteins

Plasma on DBS

Trap + 75 Pm × 150 mm (Agilent)

No – compared with WB and ELISA

Comprehensive mode Obesity

Plasma depleted 75 Pm × 150 mm with MARS (5 Pm, 200 Å) column

Diabetes

Plasma and erythrocytes

Biomarkers Plasma identification and microparticles quantification

MARS: Multiple affinity removal system; N/A: Not applicable; WB: Western blot.



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Review


Table 4. Performance of various approaches described in this review, regarding a number of factors appreciated in, for example, clinical settings. Feature

Approach

Analysis time

Reproducibility

Sensitivity

‘Plug and play’-ness

Automation

Robustness/ maintenance

Column morphology

Packed nano-LC

XX

XXX

XX

XX

XXX

XX

Add-on/ configuration

Monoliths

XX

X

XX

XX

XXX

XX

PLOT

X

XX†

XXX†

X

XXX

XX

Online SPE

XX

XXX

XXX

XX

XXX

XXX

†

IMER

XXX

N/A

XX

X

N/A

N/A

2D

X

N/A

XXX

X

X

N/A

Chip systems

XX

N/A

XXX

XXX

XXX

XX

†

Based on few studies describing reproducibility/sensitivity. IMER: Immobilized enzyme reactor; N/A: Few/no studies available describing the trait; PLOT: Polymer layer open tubular; X: Low; XX: Medium; XXX: High.

higher degree of automation, but also repeatability, which often goes hand in hand with automated approaches. However, a number of automatable approaches (e.g., temperature manipulation, or variants of enzyme reactors) with potential popularity have not been embraced by proteomics laboratories. A reason may be that they often are only described with a proof of principle with a very simple set of standards, and not followed up by application studies with biosamples; this may not be enough evidence for busy, routine labs. Therefore, we hope to see more ‘real life’ demonstrations to ingenious solutions by, for example, separation scientists/polymer chemists to awaken the appetite of proteomics people toward higher automation in nano-LC and proteomics. As discussed above, nano-LC can be robust and reproducible enough to be used for clinical applications, for example, measuring sub-ng/ml biomarkers [80] . Many nano-LC–MS users are eager to see their approaches be of aid to patients and doctors. However, many leaders of medical research facilities are skeptical (or unknowing?) to target protein measurements with nano-LC–MS, sticking to fast and less expensive ELISA methods (even though they can have significant issues and limitations) [117] . Therefore, it may be a goal for more nano-LC users and manufacturers to promote the nano-LC–MS platform through emphasis on method validity and reliability, performing full FDA analytical chemistry guideline validations for target compounds (preferably with both external and internal standards, and not just retention time prediction tools). In other words, convince the ‘outside world’ that nano-LC– MS it is not just a time-taking research tool for deep comprehensive proteomics (which is after all nanoLC–MS’ greatest achievement so far). Also, it may be a responsibility to instrument manufacturers to not oversell their nano-LC–MS product; the plat-

1810


form is without doubt a tool for expert hands, and selling it as otherwise can lead to disappointments in hospital hierarchies which can harm the entry of nano-LC–MS into clinical settings. In addition, vendors should also put far more emphasis on developing faster nano-LC systems; for example, loading and equilibration times can be exhaustingly slow for those routine users of MS in clinics, having them switch to more conventional systems, at the sacrifice of poorer sensitivity. Increased sensitivity is one of the major reasons for moving to nano-scale chromatographic systems. However, it has been claimed that sample volume/amount is not a concern in clinics [118] . This may be true today, but there may be a change when more low-abundant biomarkers are discovered and found to be decisive in diagnosis, and push toward analysis of smaller biosamples. For the next 5–10 years, the use of nano-LC in clinics will possibly be rather limited, until some of the issues have been resolved. Still, nano-LC is expected to play an even larger role in research proteomics to understand diseases and discover new biomarkers. Undoubtedly, several relevant studies have not been described in the limited space and focus of this review. We invite readers to join our review and link to deserving papers through social media with the tag #nanoLC_bioanalysis. Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.



Review

Executive summary s The key advantage of nano-LC over conventional LC is increased sensitivity, particularly for limited samples, due to, for example, narrow column inner diameter, which reduces radial dilution. s Relation between inner diameter and column efficiency is not straightforward. s Nano-LC columns are widely commercially available, but can be packed in-lab as well. s Monolithic/open tubular columns may have defined roles in proteomics: – Silica monoliths may find a ‘killer application’ in long gradient, deep proteomics profiling; – Organic monoliths may be a ticket to high-performance top-down proteomics; – Open tubular columns for very limited samples. s A number of approaches and add-ons can enhance nano-LC performance: – Solvents and temperature can enhance sensitivity and/or nano-LC performance; – A variety of precolumns for (selective) online enrichment are available; – 2D LC can improve chromatographic resolution; – Immobilized enzyme reactors can be coupled online, increase speed of sample preparation. s Nano-LC–MS is an established research tool, but needs more focus on speed, simplicity and robustness toward clinical applications.

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