A general, robust method for the quality control of intact proteins using LC–ESI-MS

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Abstract

A simple and robust method for the routine quality control of intact proteins based on liquid chromatography coupled to electrospray ionization mass spectrometry (LC–ESI-MS) is presented. A wide range of prokaryotic and eukaryotic proteins expressed recombinantly in Escherichia coli or Pichia pastoris has been analyzed with medium- to high-throughput with on-line desalting from multi-well sample plates. Particular advantages of the method include fast chromatography and short cycle times, the use of inexpensive trapping/desalting columns, low sample carryover, and the ability to analyze proteins with masses ranging from 5 to 100 kDa with greater than 50 ppm accuracy. Moreover, the method can be readily coupled with optimized chemical reduction and alkylation steps to facilitate the analysis of denatured or incorrectly folded proteins (e.g., recombinant proteins sequestered in E. coli inclusion bodies) bearing cysteine residues, which otherwise form intractable multimers and non-specific adducts by disulfide bond formation.

Introduction

Verification of the identity of recombinant proteins and engineered variants thereof is a mandatory step in all biochemical analysis, including antibody generation, enzyme kinetic studies, and structure determination. Specifically, the presence of side products, degradation products, or unwanted protein variants which can confound future utilization or analysis must be revealed. Protein expression in high-throughput, multiwell plate format presents a specific risk of unintentional cross-contamination of proteins during sample handling, due to the close proximity of the individual sample chambers. Quality control methods for the high-throughput production of proteins must likewise be free from sample-to-sample carry over, and should possess sufficient mass accuracy and resolution to reveal cross contamination or protein heterogeneity. The ability to identify single amino acid variants and low-mass post-translational modifications across a large mass range, e.g., 5–100 kDa, is desirable. Whereas denaturing polyacrylamide gel analysis has become the de facto standard to assess protein purity, this technique suffers from limited mass accuracy and resolution. Mass spectrometry (MS), on the other hand, is an excellent tool allowing highly accurate protein mass determination.

Two ionization techniques are widely available for the production of intact, gas phase protein ions: matrix assisted laser desorption ionization (MALDI) [1] and electrospray ionization (ESI) [2]. As samples are typically introduced into the mass spectrometer in an array format, MALDI–MS is particularly well-suited to high-throughput applications, and has therefore become a workhorse for the rapid analysis of peptide digests (<4 kDa). However, the use of MALDI–MS in the analysis of larger proteins is hampered by decreasing ionization efficiency with increasing mass and the production of predominantly single- or double-charged molecular ions ([M+H]+ or [M+2H]2+, respectively). Linear time-of-flight (TOF) mass analyzers used with MALDI for intact protein analysis thus have high mass-to-charge (m/z) ranges at the expense of resolution and mass accuracy, which are often insufficient to unambiguously confirm protein sequences. In contrast, ESI generates a distribution of multiply charged molecular ions ([M+nH]n+) that, in the case of monomeric proteins, typically lies in the range 500–2000 m/z. A high degree of data redundancy, coupled with the ability to accurately calibrate high resolution analyzers in this range (e.g., reflectron TOF), yields highly accurate protein masses after peak deconvolution. For example, ESI-orthogonal acceleration TOF MS has been used to achieve <5 ppm accuracy in the analysis of an intact 30 kDa glycoprotein in the presence of a 17 kDa internal protein standard [3].

The continuous nature of ESI allows for straightforward interfacing with liquid chromatography (LC), thus providing the opportunity for on-line sample concentration, desalting, and separation. Both single stream and parallel stream LC–ESI-MS configurations have been described for the high-throughput analysis of small molecules [4] and proteins up to 9 kDa [5]. A limited number of reports have been presented describing the LC–ESI-MS analysis of larger proteins, but these are limited in scope by the need for long chromatography cycles, protein-specific eluants, and costly chromatography media. For example, a LC–ESI-MS system was used for the analysis of the intact intrinsic membrane protein bacteriorhodopsin, which had an elution time of over 40 min [6]. Furthermore, no generally applicable LC–ESI-MS methods have been described for the routine mass determination of intact proteins above 10 kDa in a multi-well plate format. Motivated by demands to characterize and perform quality control analysis on a diversity of proteins expressed in microbial hosts, we have developed a rapid, robust system for MS analysis of proteins in the mass range 10–100 kDa from liquid samples, including on-line desalting. Demonstrative examples include the mass analysis of functional enzymes, heterogeneously N-glycosylated proteins, a range of native (i.e., folded and soluble) human protein targets for crystallography, and urea-solubilized human protein fragments for antibody generation. For the analysis of denatured protein samples, such as those produced from E. coli inclusion bodies, it was further shown that chemical reduction and alkylation was essential to cleave non-specific disulfide bonds to cysteine residues, improve signal quality, and simplify MS analysis.

Section snippets

Chemicals

HPLC gradient acetonitrile (ACN), was from Carlo Erba (Peypin, France). Formic acid (FA) was from Fluka Chemie Gmbh (Buchs, Germany). Dithiothreitol (DTT), iodoacetamide (IAA), and horse heart myoglobin (HHM) were from Sigma–Aldrich Chemie Gmbh (Steinheim, Germany).

Equipment

The CapLC System™ and Q-Tof ™ II quadrupole/orthogonal acceleration time-of-flight mass spectrometer were from Waters Corporation, Micromass MS Technologies (Manchester, UK). Protein trap cartridges (300 μm × 5 mm, filled with C4

Method development—analysis of HPR PrESTs

The development of the present method was motivated by the need to perform accurate quality control analysis of human protein epitope signature tags produced using high-throughput techniques by the Swedish Human Proteome Resource (http://www.proteinatlas.org/) [7], [13]. PrESTs are fragments of human proteins 25–200 amino acids in length derived from predicted open reading frames (ORFs) of the human genome, against which antibodies are raised for localization proteomics studies. As a

Conclusions

A straightforward LC–ESI-MS method is presented which is capable of analyzing intact proteins of different origin and broad mass distribution. The inherent robustness and simple configuration of the LC system, including the use of cost-effective, disposable columns and short gradient cycle times, make it suitable for use as a general method for rapid LC–ESI-MS analysis of recombinant proteins, including large and glycosylated proteins.

Acknowledgements

We thank the Knut and Alice Wallenberg Foundation (supporting the Swedish Human Proteome Resource, HPR) for funding. Purchase of the LC–MS equipment was funded by the Wallenberg Consortium North for Functional Genomics. Mathias Uhlén and Sofia Hober of the HPR and Johan Weigelt of the Structural Genomics Consortium are thanked for helpful discussions. Prof. Robert Kelly (North Carolina State University) is gratefully acknowledged for providing the T. maritima α-galactosidase expression

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