Proteomics of human prostate cancer biospecimens: the global,systems-wide perspective for Protein markers with potential clinical utility

Despite intense global efforts, no new clinical and/or viable biomarkers have been established to overcome the limitation of the prostate specific antigen in the early diagnosis and prognosis of prostate cancer (PCa). The current proteomic approaches to PCa biomarker discovery, each have distinct advantages and disadvantages, yet when combined hold real promise in the coming years. One key approach to this effort is the development of non-targeted, depletion-free and quantitative liquid chromatography–ultra high resolution tandem mass spectrometry (LC–MS) pipelines for the systems-wide interrogation of the diverse proteomes encompassed in whole tissue and blood serum or plasma. Derived quantitative proteomes can be decoded for their biomedical relevance with advanced bioinformatics and bibliographic mining to yield promising ‘molecular portraits’ that can gauge prostatic disease at the serological level. Their functional annotation, although potentially useful, is beyond our current level of biological understanding and should not be requisite for their effective use in the clinical monitoring of prostatic disease.     

High sensitivity LC-MS profiling of antibody-drug conjugates with difluoroacetic acid ion pairing

ABSTRACT

Reversed-phase liquid chromatography (RPLC) separations of proteins using optical detection generally use trifluoroacetic acid (TFA) because it is a strong, hydrophobic acid and a very effective ion-pairing agent for minimizing chromatographic secondary interactions. Conversely and in order to avoid ion suppression, analyses entailing mass spectrometry (MS) detection is often performed with a weaker ion-pairing modifier, like formic acid (FA), but resolution quality may be reduced. To gain both the chromatographic advantages of TFA and the enhanced MS sensitivity of FA, we explored the use of an alternative acid, difluoroacetic acid (DFA). This acid modifier is less acidic and less hydrophobic than TFA and is believed to advantageously affect the surface tension of electrospray droplets. Thus, it is possible to increase MS sensitivity threefold by replacing TFA with DFA. Moreover, we have observed DFA ion pairing to concomitantly produce higher chromatographic resolution than FA and even TFA. For this reason, we prepared and used MS-quality DFA in place of FA and TFA in separations involving IdeS digested, reduced NIST mAb and a proprietary antibody-drug conjugate (ADC), aiming to increase sensitivity, resolution and protein recovery. The resulting method using DFA was qualified and applied to two other ADCs and gave heightened sensitivity, resolution and protein recovery versus analyses using TFA. This new method, based on a purified, trace metal free DFA, can potentially become a state-of-the-art liquid chromatography-MS technique for the deep characterization of ADCs.     

Specific and high-resolution identification of monoclonal antibody fragments detected by capillary electrophoresis–sodium dodecyl sulfate using reversed-phase HPLC with top-down mass spectrometry analysis

ABSTRACT

In recent years, capillary electrophoresis–sodium dodecyl sulfate (cSDS) has been widely used for high resolution separation and quantification of the fragments and aggregates of monoclonal antibodies (mAbs) to ensure the quality of mAb therapeutics. However, identification of the low-molecular-weight (LMW) and high-molecular-weight (HMW) species detected in cSDS electropherograms has been based primarily on the approximate MWs calculated from standard curves using known MW standards and correlations with fragments and aggregates identified by other methods. It is not easy to collect sufficient amounts of H/LMW species from cSDS for analysis by orthogonal methods and the direct coupling of cSDS with mass spectrometry (MS) is very difficult due to interference from SDS. In this study, we describe the precise identification of H/LMW species detected by cSDS using reversed-phase high performance liquid chromatography (RP-HPLC) coupled with top-down tandem MS analysis. The H/LMW species were first identified by on-line RP-HPLC MS analysis and the RP-HPLC fractions were then analyzed by cSDS to connect the identified H/LMW species with the peaks in the cSDS electropherogram. With this method, 58 unique H/LMW species were identified from an immunoglobulin G1 (IgG1) mAb. The identified fragments ranged from 10 kDa single chain fragments to 130 kDa triple chain fragments, including some with post-translational modifications. This is the first study to clearly identify the antibody fragments, including the exact clipping sites, observed in cSDS electropherograms. The methodology and results presented here should be applicable to most other IgG1 mAbs.     

A QUANTITATIVE SAMPLER FOR SUBMERGED AQUATIC MACROPHYTES

SUMMARY

A simple rotary sampler, capable of quantitatively harvesting submerged aquatic macrophytes is described. The sampler can be operated from a boat and consists of a central rod with a specially designed cutting blade at the base, and collecting hooks to catch the cut material. The values obtained with this sampler were not significantly different (at the 95% level of probabality) from those obtained by manual cutting underwater. The rotary sampler has great advantages in terms of time, ease of positioning, and effort over hand cutting.     

A fast and reproducible method for albumin isolation and depletion from serum and cerebrospinal fluid

Analysis of serum and plasma proteomes is a common approach for biomarker discovery, and the removal of high‐abundant proteins, such as albumin and immunoglobins, is usually the first step in the analysis. However, albumin binds peptides and proteins, which raises concerns as to how the removal of albumin could impact the outcome of the biomarker study while ignoring the possibility that this could be a biomarker subproteome itself. The first goal of this study was to test a new commercially available affinity capture reagent from Protea Biosciences and to compare the efficiency and reproducibility to four other commercially available albumin depletion methods. The second goal of this study was to determine if there is a highly efficient albumin depletion/isolation system that minimizes sample handling and would be suitable for large numbers of samples. Two of the methods tested (Sigma and ProteaPrep) showed an albumin depletion efficiency of 97% or greater for both serum and cerebrospinal fluid (CSF). Isolated serum and CSF albuminomes from ProteaPrep spin columns were analyzed directly by LC‐MS/MS, identifying 128 serum (45 not previously reported) and 94 CSF albuminome proteins (17 unique to the CSF albuminome). Serum albuminome was also isolated using Vivapure anti‐HSA columns for comparison, identifying 105 proteins, 81 of which overlapped with the ProteaPrep method.     

Bottom‐up proteome analysis of E. coli using capillary zone electrophoresis‐tandem mass spectrometry with an electrokinetic sheath‐flow electrospray interface

The Escherichia coli proteome was digested with trypsin and fractionated using SPE on a C18 SPE column. Seven fractions were collected and analyzed by CZE‐ESI‐MS/MS. The separation was performed in a 60‐cm‐long linear polyacrylamide‐coated capillary with a 0.1% v/v formic acid separation buffer. An electrokinetic sheath‐flow electrospray interface was used to couple the separation capillary with an Orbitrap‐Velos operating in higher‐energy collisional dissociation mode. Each CZE‐ESI‐MS/MS run lasted 50 min and total MS time was 350 min. A total of 23 706 peptide spectra matches, 4902 peptide IDs, and 871 protein group IDs were generated using MASCOT with false discovery rate less than 1% on the peptide level. The total mass spectrometer analysis time was less than 6 h, the sample identification rate (145 proteins/h) was more than two times higher than previous studies of the E. coli proteome, and the amount of sample consumed (<1 μg) was roughly fourfold less than previous studies. These results demonstrate that CZE is a useful tool for the bottom‐up analysis of prokaryote proteomes.     

Systematic research on the pretreatment of peptides for quantitative proteomics using a C18 microcolumn

Reversed phase microcolumns have been widely used for peptide pretreatment to desalt and remove interferences before tandem LC–MS in proteomics studies. However, few studies have characterized the effects of experimental parameters as well as column characteristics on the composition of identified peptides. In this study, several parameters including the concentration of ACN in washing buffer, the microcolumn's purification effect, the peptide recovery rate, and the dynamic‐binding capacity were characterized in detail, based upon stable isotope labeling by amino acids in a cell culture quantitative approach. The results showed that peptide losses can be reduced with low ACN concentration in washing buffers resulting in a recovery rate of approximately 82%. Furthermore, the effects of ACN concentration and loading amount on the properties of identified peptides were also evaluated. We found that the dynamic‐binding capacity of the column was approximately 26 μg. With increased loading amounts, more hydrophilic peptides were replaced by hydrophobic peptides.     

Global combined precursor isotopic labeling and isobaric tagging (cPILOT) approach with selective MS3 acquisition

Recently, we reported a novel proteomics quantitation scheme termed “combined precursor isotopic labeling and isobaric tagging (cPILOT)” that allows for the identification and quantitation of nitrated peptides in as many as 12–16 samples in a single experiment. cPILOT offers enhanced multiplexing and posttranslational modification specificity, however excludes global quantitation for all peptides present in a mixture and underestimates reporter ion ratios similar to other isobaric tagging methods due to precursor co‐isolation. Here, we present a novel chemical workflow for cPILOT that can be used for global tagging of all peptides in a mixture. Specifically, through low pH precursor dimethylation of tryptic or LysC peptides followed by high pH tandem mass tags, the same reporter ion can be used twice in a single experiment. Also, to improve triple‐stage mass spectrometry (MS³) data acquisition, a selective MS³ method that focuses on product selection of the y₁ fragment of lysine‐terminated peptides is incorporated into the workflow. This novel cPILOT workflow has potential for global peptide quantitation that could lead to enhanced sample multiplexing and increase the number of quantifiable spectra obtained from MS³ acquisition methods.