Effect of difluoroacetic acid and biological matrices on the development of a liquid chromatography–triple quadrupole mass spectrometry method for determination of intact growth factor proteins

Abstract

In biological systems, variable protein expression is a crucial marker for numerous diseases, including cancer. The vast majority of liquid chromatography-triple quadrupole mass spectrometry-based quantitative protein assays use bottom-up methodologies, where proteins are subjected to proteolytic cleavage prior to analysis. Here, the effect of difluoroacetic acid and biological matrices on the developement of a multiple reaction monitoring based top-down reversed-phase liquid chromatography-triple quadrupole mass spectrometry method for analysis of cancerrelated intact proteins was evaluated. Seven growth factors (5.5-26.5 kDa; isoelectric points: 4.6-9.9) were analyzed on a wide-pore C4 column. The optimized method was performed at 30 • C, using a 0.2 mL/min flow rate, a 10 %B/min gradient slope, and 0.05% v/v difluoroacetic acid as a mobile phase modifier. The increase of mass spectrometry sensitivity due to the difluoroacetic acid (estimated limits of detection in biological matrices 1-500 ng/mL) significantly varied for proteins with lower and higher charge state distributions. Matrix effects, as well as the specificity of the method were assessed for variable biological samples and pretreatment methods. This work demonstrates method development to improve the ability to target intact proteins directly by more affordable triple quadrupole mass spectrometry instrumentation, which could be beneficial in many application fields. K E Y W O R D Sbiological fluids,

Sample preparation and fractionation techniques for intact proteins for mass spectrometric analysis

“…Titanium or stainless steel is preferred for column frits, but stainless steel and/or PEEK‐Sil do not always prevent unwanted adsorption [64]. The use of ion‐pairing reagents or surfactants in mobile phases can help reduce unwanted adsorption, which would otherwise ultimately result in greater carry‐over and losses in method performance; these should be volatile ion‐pairing reagents if MS detection via electrospray ionization (ESI) is desired [27,65,66]. Strong adsorption of proteins on the capillary walls is one of the major drawbacks also in CE.…”

“…The analysis of intact proteins can be performed by a variety of MS/MS platforms, and the ionization source(s), mass analyzer(s), and available ion activation and dissociation technique(s) composed thereof should be considered before adoption for a particular application. Proteins can be ionized by ESI [27,65,80,81] or MALDI‐MS [75,82–84] and then analyzed by QQQ [27,65,81], Q‐TOF [71–75], linear trap quadrupole (LTQ)‐Orbitrap [74,85–87], or FT‐ICR [83,88–92] mass analyzers. Recently, the ability of Orbitrap MS to assign charge states and masses to proteins and their fragment ions has been demonstrated; this is based on the relationship between the cumulative ion intensity and acquisition time of individual ions [93–95].…”

“…Recently, the ability of Orbitrap MS to assign charge states and masses to proteins and their fragment ions has been demonstrated; this is based on the relationship between the cumulative ion intensity and acquisition time of individual ions [93–95]. Fragmentation can be achieved by collision‐induced dissociation [27,65,74,83,91], higher‐energy C‐trap dissociation [86,96], infrared multiphoton dissociation [83,90], ultraviolet photodissociation [97], electron capture dissociation [83,90,92], electron‐transfer dissociation [84], or in‐source decay [75]. Collision‐induced dissociation is currently the most commonly used MS/MS fragmentation technique for top‐down proteomics, though the choice of fragmentation technique depends upon availability and the goals of the analysis.…”

“…While the primary advantage of using immunoaffinity‐based methods is high selectivity, this can also be a drawback if one desires to simultaneously analyze a large number of different proteins from a single sample. To be able to interrogate several proteins in one analysis with high accuracy, precision, and selectivity is an advantage of CE‐MS, but much less emphasis has been placed on standardizing sample preparation that can reliably target multiple proteins, which have varying physicochemical properties [27]. Accordingly, this review focuses primarily on examining the range of non‐immunoaffinity‐based methods available or under development for the preparation of intact proteins.…”

Peptide separation selectivity in proteomics LC‐MS experiments: Comparison of formic and mixed formic/heptafluorobutyric acids ion‐pairing modifiers

“…Subsequent development led to a wider use of 0.1% FA as it provided better protein identification output, even at the cost of losing hydrophilic analytes. Therefore, detection of hydrophilic peptides still represents a significant problem for modern proteomic applications [7] and is being addressed in many studies [3][4][5][7][8][9][10] including attempts to find alternative eluent additives [20,21].…”